http://measurebiology.org/w/api.php?action=feedcontributions&user=Juliesutton&feedformat=atomCourse Wiki - User contributions [en]2024-03-29T12:38:06ZUser contributionsMediaWiki 1.22.3http://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-29T19:44:53Z<p>Juliesutton: /* Growing yeast cultures */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
<br />
==YPD broth==<br />
YPD broth is delicious for yeast, but terribly auto fluorescent. For some reason that I don't understand, yeast will not pellet after centrifuging in synthetic medium, but it will in YPD. That's the only real reason we need it for the 20.309 experiment, but in principle it could be used to grow liquid cultures as well.<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth powder<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
Using the Formedium LoFlo yeast nitrogen base without riboflavin and folic acid significantly reduces the autofluorescence of the medium relative to regular yeast nitrogen base. It's also better to filter sterilize the medium, rather than autoclaving, since autoclaving also appears to make the autofluorescence worse. <br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x low fluorescence SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g LoFlo nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.<br />
# Filter sterilize using a 0.2um filtration system into sterile bottles.<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
<br />
You should find the yeast stock in a box on the top shelf of the 20.109 -80 freezer labeled "20.309 Yeast stocks". ''Note Jan 2021: I can't remember exactly how the cells are labeled. There may be two or three variants in the box. One is a purchased variant from Thermo with only the Hog1-GFP mutant, and there may be an additional Hog1-gfp only strain from the Amon lab. Look for the one that is labeled two-color, or Hog1-GFP MCP-TagRFP or something the most elaborate.'' <br />
<br />
<figure id="fig:20.309 yeast stock"><br />
[[Image:freezer stock.png|thumb|right|20.309 freezer stock.]]<br />
</figure><br />
<br />
Use an autoclaved toothpick to scrape some of the freezer stock and spread it on a YPD plate. Let grow at room temperature or at 30C for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
==Making new freezer stock==<br />
To make a new freezer stock, <br />
# grow up cells on YPD plate, <br />
# scrape up a bunch of healthy colonies with with sterile toothpick, <br />
# mix toothpick into cryo tube with YPD+30% glycerol<br />
<br />
''Note Jan 2021: I have yet to do this, but it will need to be done eventually.''<br />
<br />
==Liquid Culture==<br />
<br />
(Yeast are happiest at 30C, so most protocols will recommend growing the liquid culture at 30C. But they will still grow fine at room temperature. Since our experiments are done at room temperature, it's best to grow the cultures at room temperature, too, so you won't shock them by changing their environment right before the experiment.)<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-29T16:20:33Z<p>Juliesutton: /* Plate */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
<br />
==YPD broth==<br />
YPD broth is delicious for yeast, but terribly auto fluorescent. For some reason that I don't understand, yeast will not pellet after centrifuging in synthetic medium, but it will in YPD. That's the only real reason we need it for the 20.309 experiment, but in principle it could be used to grow liquid cultures as well.<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth powder<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
Using the Formedium LoFlo yeast nitrogen base without riboflavin and folic acid significantly reduces the autofluorescence of the medium relative to regular yeast nitrogen base. It's also better to filter sterilize the medium, rather than autoclaving, since autoclaving also appears to make the autofluorescence worse. <br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x low fluorescence SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g LoFlo nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.<br />
# Filter sterilize using a 0.2um filtration system into sterile bottles.<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
<br />
You should find the yeast stock in a box on the top shelf of the 20.109 -80 freezer labeled "20.309 Yeast stocks". ''Note Jan 2021: I can't remember exactly how the cells are labeled. There may be two or three variants in the box. One is a purchased variant from Thermo with only the Hog1-GFP mutant, and there may be an additional Hog1-gfp only strain from the Amon lab. Look for the one that is labeled two-color, or Hog1-GFP MCP-TagRFP or something the most elaborate.'' <br />
<br />
<figure id="fig:20.309 yeast stock"><br />
[[Image:freezer stock.png|thumb|right|20.309 freezer stock.]]<br />
</figure><br />
<br />
Use an autoclaved toothpick to scrape some of the freezer stock and spread it on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
==Making new freezer stock==<br />
To make a new freezer stock, <br />
# grow up cells on YPD plate, <br />
# scrape up a bunch of healthy colonies with with sterile toothpick, <br />
# mix toothpick into cryo tube with YPD+30% glycerol<br />
<br />
''Note Jan 2021: I have yet to do this, but it will need to be done eventually.''<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/File:Freezer_stock.pngFile:Freezer stock.png2021-01-29T16:19:55Z<p>Juliesutton: </p>
<hr />
<div></div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-29T16:19:32Z<p>Juliesutton: /* Growing yeast cultures */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
<br />
==YPD broth==<br />
YPD broth is delicious for yeast, but terribly auto fluorescent. For some reason that I don't understand, yeast will not pellet after centrifuging in synthetic medium, but it will in YPD. That's the only real reason we need it for the 20.309 experiment, but in principle it could be used to grow liquid cultures as well.<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth powder<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
Using the Formedium LoFlo yeast nitrogen base without riboflavin and folic acid significantly reduces the autofluorescence of the medium relative to regular yeast nitrogen base. It's also better to filter sterilize the medium, rather than autoclaving, since autoclaving also appears to make the autofluorescence worse. <br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x low fluorescence SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g LoFlo nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.<br />
# Filter sterilize using a 0.2um filtration system into sterile bottles.<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
<br />
You should find the yeast stock in a box on the top shelf of the 20.109 -80 freezer labeled "20.309 Yeast stocks". ''Note Jan 2021: I can't remember exactly how the cells are labeled. There may be two or three variants in the box. One is a purchased variant from Thermo with only the Hog1-GFP mutant, and there may be an additional Hog1-gfp only strain from the Amon lab. Look for the one that is labeled two-color, or Hog1-GFP MCP-TagRFP or something the most elaborate.'' <br />
<br />
<figure id="fig:20.309 yeast stock"><br />
|[[Image:freezer stock.png|thumb|right|20.309 freezer stock.]]<br />
</figure><br />
<br />
Use an autoclaved toothpick to scrape some of the freezer stock and spread it on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
<br />
==Making new freezer stock==<br />
To make a new freezer stock, <br />
# grow up cells on YPD plate, <br />
# scrape up a bunch of healthy colonies with with sterile toothpick, <br />
# mix toothpick into cryo tube with YPD+30% glycerol<br />
<br />
''Note Jan 2021: I have yet to do this, but it will need to be done eventually.''<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-29T16:16:30Z<p>Juliesutton: /* Making new freezer stock */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
<br />
==YPD broth==<br />
YPD broth is delicious for yeast, but terribly auto fluorescent. For some reason that I don't understand, yeast will not pellet after centrifuging in synthetic medium, but it will in YPD. That's the only real reason we need it for the 20.309 experiment, but in principle it could be used to grow liquid cultures as well.<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth powder<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
Using the Formedium LoFlo yeast nitrogen base without riboflavin and folic acid significantly reduces the autofluorescence of the medium relative to regular yeast nitrogen base. It's also better to filter sterilize the medium, rather than autoclaving, since autoclaving also appears to make the autofluorescence worse. <br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x low fluorescence SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g LoFlo nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.<br />
# Filter sterilize using a 0.2um filtration system into sterile bottles.<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
<br />
You should find the yeast stock in a box on the top shelf of the 20.109 -80 freezer labeled "20.309 Yeast stocks". ''Note Jan 2021: I can't remember exactly how the cells are labeled. There may be two or three variants in the box. One is a purchased variant from Thermo with only the Hog1-GFP mutant, and there may be an additional Hog1-gfp only strain from the Amon lab. Look for the one that is labeled two-color, or Hog1-GFP MCP-TagRFP or something the most elaborate'' <br />
<br />
Use an autoclaved toothpick to scrape some of the freezer stock and spread it on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
<br />
==Making new freezer stock==<br />
To make a new freezer stock, <br />
# grow up cells on YPD plate, <br />
# scrape up a bunch of healthy colonies with with sterile toothpick, <br />
# mix toothpick into cryo tube with YPD+30% glycerol<br />
<br />
''Note Jan 2021: I have yet to do this, but it will need to be done eventually.''<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-29T16:15:49Z<p>Juliesutton: /* Making new freezer stock */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
<br />
==YPD broth==<br />
YPD broth is delicious for yeast, but terribly auto fluorescent. For some reason that I don't understand, yeast will not pellet after centrifuging in synthetic medium, but it will in YPD. That's the only real reason we need it for the 20.309 experiment, but in principle it could be used to grow liquid cultures as well.<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth powder<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
Using the Formedium LoFlo yeast nitrogen base without riboflavin and folic acid significantly reduces the autofluorescence of the medium relative to regular yeast nitrogen base. It's also better to filter sterilize the medium, rather than autoclaving, since autoclaving also appears to make the autofluorescence worse. <br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x low fluorescence SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g LoFlo nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.<br />
# Filter sterilize using a 0.2um filtration system into sterile bottles.<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
<br />
You should find the yeast stock in a box on the top shelf of the 20.109 -80 freezer labeled "20.309 Yeast stocks". ''Note Jan 2021: I can't remember exactly how the cells are labeled. There may be two or three variants in the box. One is a purchased variant from Thermo with only the Hog1-GFP mutant, and there may be an additional Hog1-gfp only strain from the Amon lab. Look for the one that is labeled two-color, or Hog1-GFP MCP-TagRFP or something the most elaborate'' <br />
<br />
Use an autoclaved toothpick to scrape some of the freezer stock and spread it on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
<br />
==Making new freezer stock==<br />
To make a new freezer stock, <br />
# grow up cells on YPD plate, <br />
# scrape up a bunch of healthy colonies with with sterile toothpick, <br />
# mix toothpick into cryo tube with YPD+30% glycerol<br />
<br />
(note: I have yet to do this, but it will need to be done eventually.)<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-28T22:28:57Z<p>Juliesutton: /* Growing yeast cultures */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
<br />
==YPD broth==<br />
YPD broth is delicious for yeast, but terribly auto fluorescent. For some reason that I don't understand, yeast will not pellet after centrifuging in synthetic medium, but it will in YPD. That's the only real reason we need it for the 20.309 experiment, but in principle it could be used to grow liquid cultures as well.<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth powder<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
Using the Formedium LoFlo yeast nitrogen base without riboflavin and folic acid significantly reduces the autofluorescence of the medium relative to regular yeast nitrogen base. It's also better to filter sterilize the medium, rather than autoclaving, since autoclaving also appears to make the autofluorescence worse. <br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x low fluorescence SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g LoFlo nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.<br />
# Filter sterilize using a 0.2um filtration system into sterile bottles.<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
<br />
You should find the yeast stock in a box on the top shelf of the 20.109 -80 freezer labeled "20.309 Yeast stocks". ''Note Jan 2021: I can't remember exactly how the cells are labeled. There may be two or three variants in the box. One is a purchased variant from Thermo with only the Hog1-GFP mutant, and there may be an additional Hog1-gfp only strain from the Amon lab. Look for the one that is labeled two-color, or Hog1-GFP MCP-TagRFP or something the most elaborate'' <br />
<br />
Use an autoclaved toothpick to scrape some of the freezer stock and spread it on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
<br />
==Making new freezer stock==<br />
To make a new freezer stock, <br />
# grow up cells on YPD plate, <br />
# scrape up a whole bunch of colonies with with sterile toothpick, <br />
# mix toothpick into cryo tube with YPD+30% glycerol<br />
<br />
(note: I have yet to do this, but it will need to be done eventually.)<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-28T22:22:57Z<p>Juliesutton: /* Growing yeast cultures */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
<br />
==YPD broth==<br />
YPD broth is delicious for yeast, but terribly auto fluorescent. For some reason that I don't understand, yeast will not pellet after centrifuging in synthetic medium, but it will in YPD. That's the only real reason we need it for the 20.309 experiment, but in principle it could be used to grow liquid cultures as well.<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth powder<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
Using the Formedium LoFlo yeast nitrogen base without riboflavin and folic acid significantly reduces the autofluorescence of the medium relative to regular yeast nitrogen base. It's also better to filter sterilize the medium, rather than autoclaving, since autoclaving also appears to make the autofluorescence worse. <br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x low fluorescence SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g LoFlo nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.<br />
# Filter sterilize using a 0.2um filtration system into sterile bottles.<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
Use an autoclaved toothpick to scrape some of the freezer stock. Spread on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
<br />
==Making new freezer stock==<br />
To make a new freezer stock, <br />
# grow up cells on YPD plate, <br />
# scrape up a whole bunch of colonies with with sterile toothpick, <br />
# mix toothpick into cryo tube with YPD+30% glycerol<br />
<br />
(note: I have yet to do this, but it will need to be done eventually.)<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-28T22:17:21Z<p>Juliesutton: /* Protocols */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
<br />
==YPD broth==<br />
YPD broth is delicious for yeast, but terribly auto fluorescent. For some reason that I don't understand, yeast will not pellet after centrifuging in synthetic medium, but it will in YPD. That's the only real reason we need it for the 20.309 experiment, but in principle it could be used to grow liquid cultures as well.<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth powder<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
Using the Formedium LoFlo yeast nitrogen base without riboflavin and folic acid significantly reduces the autofluorescence of the medium relative to regular yeast nitrogen base. It's also better to filter sterilize the medium, rather than autoclaving, since autoclaving also appears to make the autofluorescence worse. <br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x low fluorescence SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g LoFlo nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.<br />
# Filter sterilize using a 0.2um filtration system into sterile bottles.<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
Use an autoclaved toothpick to scrape some of the freezer stock. Spread on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-28T22:16:39Z<p>Juliesutton: /* YPD broth */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
Using the Formedium LoFlo yeast nitrogen base without riboflavin and folic acid significantly reduces the autofluorescence of the medium relative to regular yeast nitrogen base. It's also better to filter sterilize the medium, rather than autoclaving, since autoclaving also appears to make the autofluorescence worse. <br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x low fluorescence SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g LoFlo nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.<br />
# Filter sterilize using a 0.2um filtration system into sterile bottles.<br />
<br />
==YPD broth==<br />
YPD broth is delicious for yeast, but terribly auto fluorescent. For some reason that I don't understand, yeast will not pellet after centrifuging in synthetic medium, but it will in YPD. That's the only real reason we need it for the 20.309 experiment, but in principle it could be used to grow liquid cultures as well.<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth powder<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
Use an autoclaved toothpick to scrape some of the freezer stock. Spread on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-28T22:14:08Z<p>Juliesutton: /* YPD plates */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
Using the Formedium LoFlo yeast nitrogen base without riboflavin and folic acid significantly reduces the autofluorescence of the medium relative to regular yeast nitrogen base. It's also better to filter sterilize the medium, rather than autoclaving, since autoclaving also appears to make the autofluorescence worse. <br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x low fluorescence SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g LoFlo nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.<br />
# Filter sterilize using a 0.2um filtration system into sterile bottles.<br />
<br />
==YPD broth==<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth powder<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
Use an autoclaved toothpick to scrape some of the freezer stock. Spread on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-28T22:13:50Z<p>Juliesutton: /* For 500 mL each of low and high salt medium */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
Using the Formedium LoFlo yeast nitrogen base without riboflavin and folic acid significantly reduces the autofluorescence of the medium relative to regular yeast nitrogen base. It's also better to filter sterilize the medium, rather than autoclaving, since autoclaving also appears to make the autofluorescence worse. <br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x low fluorescence SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g LoFlo nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.<br />
# Filter sterilize using a 0.2um filtration system into sterile bottles.<br />
<br />
==YPD broth==<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
Use an autoclaved toothpick to scrape some of the freezer stock. Spread on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-28T22:12:39Z<p>Juliesutton: /* Synthetic Complete culture media */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
Using the Formedium LoFlo yeast nitrogen base without riboflavin and folic acid significantly reduces the autofluorescence of the medium relative to regular yeast nitrogen base. It's also better to filter sterilize the medium, rather than autoclaving, since autoclaving also appears to make the autofluorescence worse. <br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x low fluorescence SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g LoFlo nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.<br />
# Filter sterilize using a 0.2um filtration system into sterile bottles.<br />
<br />
# Follow the same steps to make an extra bottle of low salt medium for culturing purposes, if desired.<br />
<br />
==YPD broth==<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
Use an autoclaved toothpick to scrape some of the freezer stock. Spread on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-28T22:09:19Z<p>Juliesutton: /* Synthetic Complete culture media */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
Using the Formedium LoFlo yeast nitrogen base without riboflavin and folic acid significantly reduces the autofluorescence of the medium relative to regular yeast nitrogen base. It's also better to filter sterilize the medium, rather than autoclaving, since autoclaving also appears to make the autofluorescence worse. <br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x low fluorescence SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g LoFlo nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.<br />
# Filter sterilize using a 0.2um filtration system into sterile bottles.<br />
<br />
==YPD broth==<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
Use an autoclaved toothpick to scrape some of the freezer stock. Spread on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-28T18:41:41Z<p>Juliesutton: /* Liquid Culture */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water.<br />
# Autoclave both bottles, as well as 50% glucose stock for 25 minutes.<br />
# Use a sterile pipette to add 20 ml of 50% glucose to each bottle.<br />
<br />
''Edit Fa2019: filter sterilize rather than autoclaving ''<br />
<br />
==YPD broth==<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
Use an autoclaved toothpick to scrape some of the freezer stock. Spread on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their autofluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-28T18:39:28Z<p>Juliesutton: /* Plate */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water.<br />
# Autoclave both bottles, as well as 50% glucose stock for 25 minutes.<br />
# Use a sterile pipette to add 20 ml of 50% glucose to each bottle.<br />
<br />
''Edit Fa2019: filter sterilize rather than autoclaving ''<br />
<br />
==YPD broth==<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
Use an autoclaved toothpick to scrape some of the freezer stock. Spread on a YPD plate. Let grow at room temperature or at 30C (or room temperature) for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their fluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-28T15:04:02Z<p>Juliesutton: /* 50% Glucose Stock Solution */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water.<br />
# Autoclave both bottles, as well as 50% glucose stock for 25 minutes.<br />
# Use a sterile pipette to add 20 ml of 50% glucose to each bottle.<br />
<br />
''Edit Fa2019: filter sterilize rather than autoclaving ''<br />
<br />
==YPD broth==<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% (w/v) glucose. Because the solution is so concentrated, you can't just mix 100g H2O + 100g glucose because there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final desired volume (including stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
<br />
For a 200ml stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
Use an autoclaved toothpick to scrape some of the freezer stock. Spread on a YPD plates. Let grow at room temperature or at 30C for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their fluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-28T15:01:22Z<p>Juliesutton: /* 50% Glucose Stock Solution */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water.<br />
# Autoclave both bottles, as well as 50% glucose stock for 25 minutes.<br />
# Use a sterile pipette to add 20 ml of 50% glucose to each bottle.<br />
<br />
''Edit Fa2019: filter sterilize rather than autoclaving ''<br />
<br />
==YPD broth==<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% glucose. Because the solution is so concentrated, there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final volume (including a stir bar) on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
For a 200mM stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
Use an autoclaved toothpick to scrape some of the freezer stock. Spread on a YPD plates. Let grow at room temperature or at 30C for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their fluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Two-color_Yeast_ProtocolsTwo-color Yeast Protocols2021-01-28T15:00:12Z<p>Juliesutton: /* 50% Glucose Stock Solution */</p>
<hr />
<div>=Reagents=<br />
<br />
All part numbers are from Millipore-Sigma unless stated otherwise<br />
<br />
==YPD Broth and plates==<br />
* YPD Broth, Y1375-250G <br />
* Agar<br />
<br />
==Synthetic Complete (SC) medium==<br />
* LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from [https://www.formedium.com/us/product/yeast-nitrogen-base-without-amino-acids-and-without-folic-acid-and-riboflavin-loflo| formedium.com]<br />
* Yeast synthetic dropout powder, Y1751-20G <br />
* Histidine, H8000-5G<br />
* Glucose<br />
* NaCl<br />
<br />
==Concanavalin A==<br />
* Concanavalin A type IV, C2010-100MG<br />
* PBS<br />
<br />
=Protocols=<br />
<br />
==Concanavalin A==<br />
<br />
Make 250 uL aliquots of 1mg/ml in PBS. <br />
# Dissolve 5 mg ConA in 5 mL PBS.<br />
# Aliquot into 20x 250 uL. <br />
# Store at -20C.<br />
<br />
== Synthetic Complete culture media ==<br />
<br />
I like to make a batch of 2x stock SC medium, then divide it into three bottles:<br />
# "low salt" medium, which is the same as regular SC medium, to be used for flow experiments <br />
# "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments <br />
# the remaining SC medium I use for yeast culture.<br />
We typically need more regular medium than high salt because it is used both for flow experiments and for culturing. But I want to make sure the "high" and "low" media are prepared the exact same way so that the backgrounds match when recording images. <br />
<br />
===For 750 mL of 2 x SC stock ===<br />
# In a 1L bottle, mix the following:<br />
#* 2.88 g Drop out powder (-His), <br />
#* 114 mg histidine, <br />
#* 10.05 g nitrogen base<br />
#* 750 mL water<br />
<br />
===For 500 mL each of low and high salt medium===<br />
# Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.<br />
# Repeat step 1 for a second bottle and label it "low salt" medium.<br />
# For the low salt medium, <br />
#* Measure out 100 mL of DI water and pour into bottle.<br />
# For high salt medium, <br />
#* Measure out 100 mL of 1M NaCl and pour into bottle.<br />
# Top up each bottle with 130 mL of water.<br />
# Autoclave both bottles, as well as 50% glucose stock for 25 minutes.<br />
# Use a sterile pipette to add 20 ml of 50% glucose to each bottle.<br />
<br />
''Edit Fa2019: filter sterilize rather than autoclaving ''<br />
<br />
==YPD broth==<br />
<br />
# Mix 12.5g YPD broth powder (from sigma) with 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
<br />
== YPD plates ==<br />
<br />
# For 10 plates, mix<br />
#* 12.5g YPD broth<br />
#* 3.75g agarose<br />
#* 250 mL water.<br />
# Autoclave for 25 minutes at 121 C<br />
# When cool enough to handle, pour ~25mL of YPD-agar into each plate<br />
<br />
==50% Glucose Stock Solution==<br />
Glucose MW = 180.16 g/mol<br />
<br />
Each 1L of SC medium requires 40 ml of 50% glucose. Because the solution is so concentrated, there will be a significant volume change when mixing in the solute to solvent. My strategy is to mark the height of the final volume on the side of the bottle, then top up the solution to this volume once the bulk of the glucose is dissolved. I may be over thinking it...<br />
For a 200mM stock solution:<br />
# Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution. <br />
# Add a stir bar to the bottle and mark the water level with a piece of tape.<br />
# Pour out 1/2 of the water, leaving the stir bar behind.<br />
# Add 100g glucose to the bottle while stirring to dissolve. <br />
# When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.<br />
# When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.<br />
<br />
== 1M NaCl Stock solution ==<br />
NaCl MW = 58.44g/mol<br />
<br />
* Dissolve 29.22 g NaCl in 500mL H2O<br />
<br />
=Growing yeast cultures=<br />
<br />
==Plate==<br />
Use an autoclaved toothpick to scrape some of the freezer stock. Spread on a YPD plates. Let grow at room temperature or at 30C for 1-2 days. Once the colonies are growing, store plate in the fridge. <br />
<br />
The plates will last in the fridge for several weeks, so I typically grow one up a week or two before they are needed and keep using the plate through the semester. If the colony is overgrown or turns pink, use a sterile toothpick to spread to an empty spot on the plate.<br />
<br />
==Liquid Culture==<br />
<br />
The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary.<br />
''Fall 2019: inoculated directly from plate each day for consistency. If you inoculate at 4pm, the OD 600 the next day was typically only about 0.3 the next morning. Consider inoculating around noon to get closer to OD 600 = 1 the next morning.''<br />
<br />
# Add 10 mL of SC medium to a 125mL flask.<br />
# Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.<br />
# Submerge the stick into the culture medium and rub it against the inner wall of the flask. You should see a whitish film of cells fall off into the medium. <br />
# Attach the flask to the orbital shaker and shake at 180 rpm overnight. <br />
#* Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.<br />
#* The yeast will double in concentration about every 2.5 hours. It may be slower at the beginning, especially if the plate was in the fridge. Monitor the concentration by measuring the absorption at 600 nm. The yeast is in log phase between OD 0.4 and 0.8, and is tending towards saturated above OD = 1.<br />
#* The strain we have has a -ade mutation. My understanding is that some pathway is activated if the yeast is starved of adenine, which can happen if the cells are left saturated for too long. You can tell that this has happened if the cells look pink or red. Avoid letting the cells get to this point since it messes with their fluorescence. <br />
<br />
The day before the experiment:<br />
# Check the OD and dilute yeast into 10 ml of SC aiming for OD600 = 1 at 9am the next morning. (might need to experiment with this a bit)<br />
# At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.<br />
# Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.<br />
# At 7pm, dilute saturated culture for next day to repeat.<br />
<br />
<br />
{|class="wikitable"<br />
|+ Target yeast concentrations<br />
|- <br />
| Time<br />
| Culture 1<br />
| Culture 2<br />
| Culture 3<br />
|-<br />
| 1 pm<br />
| 0.4 <br />
|<br />
|<br />
|-<br />
| 3:30 pm<br />
| 0.8<br />
| 0.4 <br />
|<br />
|-<br />
| 6 pm<br />
|<br />
| 0.8<br />
| 0.4<br />
|-<br />
|8:30 pm <br />
|<br />
|<br />
|0.8<br />
|}<br />
<br />
Plan to use culture 1 from 1-4pm, and culture 2 from 5-7pm. Only 3 groups can use the first culture (we need 2.5ml for the dilution). Technically 5 groups can use the second culture (limit it to 4, though). That maxes us out at 7 groups per day.</div>Juliesuttonhttp://measurebiology.org/wiki/Spring_2020_Assignment_10Spring 2020 Assignment 102020-05-06T13:57:45Z<p>Juliesutton: </p>
<hr />
<div>In this assignment, you will find the amplitude and phase response of Hog1 to osmotic shock using student data from last semester. Note that this assignment is OPTIONAL, and completing it will give you +2 points of extra credit on a previous assignment. <br />
<br />
# Start by downloading the file <tt>fall2019_StudentData_Responses.mat</tt> from the [https://www.dropbox.com/sh/33d8t5a7yhhlv77/AAAerwjGLrE_sr6TjaXS764Ga/Assignment%2010?dl=0&subfolder_nav_tracking=1 course dropbox], and load the data into matlab. You should see that this file contains two variables: <br />
#* <tt>allClassResponseData</tt> is a cell array containing the analyzed response data from each group. Each element of the cell array is a 3-column matrix containing the response data. The columns represent (in order): the time (in seconds), the mean response (the average correlation between GFP and RFP in each cell), and the standard error of the response. To access the matrix of the <tt>ii</tt>th group, you can use curly braces: <tt>allClassResponseData{ii}</tt>.<br />
#* <tt>allClassOscillationPeriods</tt> is a vector containing the oscillation period (in seconds) for each of the elements in <tt>allClassResponseData</tt>.<br />
# For each set of data in <tt> allClassResponseData</tt>, use <tt>nlinfit</tt> to fit a sine function to extract the amplitude and phase of the response at that input frequency.<br />
#* Remember that <tt>nlinfit</tt> requires four inputs in this order: the independent variable (time), the dependent variable (hog1 response), a model function (more about this below), and a vector containing initial guesses for each parameter.<br />
#* Your model function should be a sine wave with an amplitude, phase, and offset as fit parameters. The offset is required because the data is not centered around zero. Additionally, you may consider adding a decreasing slope to your offset (i.e. offset = A – Bt) to help correct for some experimental artifacts (like the cells going out of focus). The frequency of oscillations of interest is determined by our experiment (given by <tt>1./allClassOscillationPeriods</tt>), and should be fixed rather than left as a fit parameter.<br />
# Plot the best-fit amplitude and phase in a Bode plot.<br />
<br />
''Tip #1:'' Test your code for a single data set before looping through the entire cell array.<br />
<br />
''Tip #2:'' The model function used by <tt>nlinfit</tt> needs to have the vector of parameters as its first argument, and the independent variable as its second argument. You may find the following template useful for the model function:<br />
<br />
<pre><br />
exampleModelFunction = @(p,t) p(1) * t.^2 + p(2) * t + p(3) t^3;<br />
bestFitParameters = nlinfit( time, response, exampleModelFunction, [1 2 3]);<br />
</pre><br />
<br />
This is an example of the model function <math>y = a t^2 + b t +c</math>, where ''a, b,'' and ''c'' are fit parameters with initial guesses a = 1, b = 2, and c =3.<br />
<br />
{{Template:Assignment Turn In|message = <br />
<br />
# Write a function to fit the Hog1 response to a sinusoid and extract the estimated amplitude and phase shift of the response signal. For each set of response data, make a plot showing the Hog1 response vs time and the best fit model function. <br />
# Make a Bode plot (i.e. plot the amplitude and phase) of the Hog1-response as a function of frequency. Use log-log axes for the magnitude, and semi-log x axes for the phase. Since some frequencies have several measurements, you may either plot each fit result as a single point on the plot, or average the results together. For the phase plot, use <tt>wrapTo180</tt> or <tt>wrapToPi</tt> to make sure your phase is in the appropriate range of angles. <br />
# Qualitatively compare your Bode plot to the amplitude and phase found in the Mettetal paper. What aspects of the results are expected or unexpected? If your measurements do not agree, describe two or three reasons why they might differ.<br />
}}<br />
<br />
{{Template:Assignment Turn In|message = Turn in all your MATLAB code in pdf format. No need to include functions that you used but did not modify.}}</div>Juliesuttonhttp://measurebiology.org/wiki/20.309_Main_Page20.309 Main Page2020-05-05T14:56:21Z<p>Juliesutton: /* Module 2: electronics, signals and systems */</p>
<hr />
<div>{{Template:20.309}}<br />
__NOTOC__<br />
Bioinstrumentation scholars: you are welcome to edit and improve this course wiki. Correct, helpful, exquisitely-expressed changes will increase your class participation grade. Alterations not meeting those critera may have the opposite effect.<br />
<br />
If you would like to contribute to this site, please [[Special:RequestAccount|request an account]].<br />
<br />
== 20.309 course information==<br />
<br />
* [[20.309:Course Information| 20.309 course information]]<br />
* [[20.309:Safety| lab safety]]<br />
* Reserve a lab station here: [http://measurebiology.org/reservations/Web/schedule.php Bioinstrumentation Lab reservation system]<br />
<br />
== 20.309 master schedule==<br />
<br />
<div style="padding: 10px; width: 740px; border: 5px solid #000FFF;"><br />
===Module 1: Optics & Microscopy===<br />
{| border=1px<br />
|style="width: 10%;"|'''Important dates''' <br />
|style="width: 15%;"|'''Assignment due'''<br />
|style="width: 30%;"|'''Assignment page links'''<br />
|style="width: 45%;"|'''Useful resources'''<br />
|-- <br />
| <font color=#04B45F>2/16/20</font color><br />
| Assignment 1<br />
| <br />
[[Assignment 1 Overview: Transillumination microscopy| Assignment 1 Overview]]<br />
* [[Assignment 1, Part 1: Pre-lab questions|Part 1: Pre-lab questions]]<br />
* [[Assignment 1, Part 2: Optics bootcamp|Part 2: Optics bootcamp]]<br />
* [[Assignment 1, Part 3: Building your transillumination microscope|Part 3: Build a microscope]]<br />
* [[Assignment 1, Part 4: Building your transilluminated microscope|Part 4: Measure stuff]]<br />
| <br />
* [https://stellar.mit.edu/S/course/20/sp20/20.309/materials.html Lectures 1 through 9 of the 20.309 class]<br />
* [http://www.microscopyu.com/articles/formulas/formulasri.html Snell's law]<br />
* [[Understanding log plots]]<br />
|-<br />
| <font color=#04B45F>2/23/20</font color><br />
| Assignment 2<br />
| [[Assignment 2: fluorescence microscopy| Assignment 2 Overview]]<br />
* [[Assignment 2 Part 1: Noise in images|Part 1: Noise in images]]<br />
* [[Assignment 2 Part 2: Fluorescence microscopy|Part 2: Fluorescence microscopy]]<br />
* [[Assignment 2 Part 3: Build an epi-illuminator for your microscope|Part 3: Build an epi-illuminator for your microscope]]<br />
|<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [[Optical aberrations]]<br />
* [[Aperture and field stops]]<br />
* [[Optical detectors, noise, and the limit of detection]]<br />
* [[Manta G032 camera measurements]]<br />
|-<br />
| <font color=#04B45F>2/25/20</font color><br />
| Quiz 1 (in lecture)<br />
| ||<br />
|-<br />
| <font color=#04B45F>3/1/20</font color><br />
| Assignment 3<br />
| [[Assignment 3 Overview|Assignment 3 Overview]]<br />
* [[Assignment 3, Part 1: visualizing actin with fluorescence contrast|Part 1: fluorescence imaging]] <br />
* [[Assignment 3, Part 2: experimental design with fluorescence| Part 2: choosing filters]] <br />
|<br />
*[[Flat-field correction|Flat-field correction of non-uniform illumination]]<br />
*[[Matlab: Scalebars]]<br />
|-<br />
| <font color=#04B45F>3/8/20</font color><br />
| Assignment 4<br />
|{{Template:Assignment 4 navigation}}<br />
|<br />
* [http://www.microscopyu.com/articles/formulas/formulasresolution.html Resolution]<br />
* [[Physical optics and resolution]]<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[MATLAB: Estimating resolution from a PSF slide image]]<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
|-<br />
| <font color=#04B45F>4/3/20</font color><br />
| Assignment 5<br />
| [[Assignment 5: Spring 2020|Assignment 5 Overview (Spring 2020)]]<br />
* [[Assignment 5, Part 1: MSD difference tracking and microscope stability|Part 1: MSD difference tracking and microscope stability]] <br />
* [[Assignment 5, Part 2: live cell particle tracking of endocytosed beads| Part 2: Live cell particle tracking of endocytosed beads]]<br />
|<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
|-<br />
| <font color=#04B45F>4/7/20</font color><br />
| Exam 1 <br />
| <br />
|<br />
|-<br />
|}<br />
<br /><br />
<br />
===Module 2: electronics, signals and systems===<br />
{| border=1px<br />
|style="width: 10%;"|'''Important dates''' <br />
|style="width: 15%;"|'''Assignment due'''<br />
|style="width: 30%;"|'''Assignment page links'''<br />
|style="width: 45%;"|'''Useful resources'''<br />
|-- <br />
| <font color=#04B45F>4/13/20</font color><br />
| Assignment 6<br />
| [[Spring 2020 Assignment 6]]<br />
* (Previous semesters' assignment 6 for reference)<br />
* [[Assignment 6 Overview: two color microscope|Assignment 6 overview]]<br />
* [[Assignment 6, Part 1: build a two-color microscope]]<br />
* [[Assignment 6, Part 2: electronics written problems]]<br />
|<br />
* [[Electronics primer]]<br />
* [https://www.khanacademy.org/science/electrical-engineering/ee-circuit-analysis-topic Khan Academy Material, esp. "Ideal Circuit Elements" and "Voltage Divider"]<br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Input and output impedance]]<br />
|-<br />
| <font color=#04B45F>4/20/20</font color><br />
| Assignment 7<br />
| [[Spring 2020 Assignment 7]]<br />
* (Previous semesters' assignment 7 for reference)<br />
* [[Electronics boot camp I: passive circuits and transfer functions|Assignment 7 overview]]<br />
* [[Electronics written problems II|Assignment 7, Part 1: more electronics written problems]]<br />
* [[Electronics boot camp lab part 1|Assignment 7, Part 2: Electronics bootcamp I]]<br />
<br />
| <br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Bode plots|Transfer functions and Bode plots]]<br />
* [[Complex Number Review|Complex number review]]<br />
* [[Input and output impedance]]<br />
|-<br />
| <font color=#04B45F>4/21/20</font color><br />
| Quiz 2 (60 min)<br />
| <br />
|<br />
|-<br />
| <font color=#04B45F>4/27/20</font color><br />
| Assignment 8<br />
| [[Spring 2020 Assignment 8]]<br />
*(Previous semester's assignment 8 for reference)<br />
*[[Assignment 8 Overview: flow channel & two-color microscope|Assignment 8 Overview]]<br />
* [[Electronics bootcamp II: feedback systems|Part 1: feedback systems]]<br />
* [[Assignment 8, Part 2: fabricate a microfluidic device| Part 2: fabricate a microfluidic device]]<br />
* [[Assignment 8, Part 3: add flow control and test your device| Part 3: add flow control and test your device]]<br />
|[http://science.sciencemag.org/content/319/5862/482 *The* yeast paper: Mettelal ''et al.'', ''Science'' (2008)]<br />
|-<br />
| <font color=#04B45F>5/4/20</font color><br />
| Assignment 9<br />
| [[Spring 2020 Assignment 9]]<br />
*(Previous semester's assignment 9 for reference)<br />
* [[Assignment 9 Overview: Analyzing yeast images]]<br />
* [[Assignment 8, Part 0: convolution practice| Part 0: convolution practice]]<br />
* [[Assignment 9, Part 1: Analyze two-color yeast images| Part 1: Analyze two-color yeast images]]<br />
* [[Assignment 10, Part 1: Measuring the osmotic shock response of yeast| A10: Measure the osmotic shock response of yeast]]<br />
|<br />
|-<br />
| <font color=#04B45F>5/8/20</font color><br />
| Exam 2 (90 min)<br />
| <br />
|<br />
|-<br />
| <font color=#04B45F>5/12/20</font color><br />
| Assignment 10 <br />
| [[Spring 2020 Assignment 10]]<br />
*(Previous semester's assignment 10 was combined with assignment 9)<br />
|<br />
|-<br />
| cancelled<br />
| Assignment 11<br />
| [[Assignment 11: Tell us about your lab visit]]<br />
| <br />
|-<br />
|}<br />
</div><br />
<br />
==Optics and microscopy resources==<br />
===Background reading===<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [[Optical aberrations]]<br />
* [[Aperture and field stops]]<br />
* [[Optical detectors, noise, and the limit of detection]]<br />
* [[Manta G032 camera measurements]]<br />
* [[Understanding log plots]]<br />
<br />
===Code examples and simulations===<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[MATLAB: Estimating resolution from a PSF slide image]]<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
<br />
==Electronics resources==<br />
===Background reading===<br />
* [[Electronics primer]]<br />
* [https://www.khanacademy.org/science/electrical-engineering/ee-circuit-analysis-topic Khan Academy Material, esp. "Ideal Circuit Elements" and "Voltage Divider"]<br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Bode plots|Transfer functions and Bode plots]]<br />
* [[Complex Number Review|Complex number review]]<br />
* [[Input and output impedance]]<br />
<br />
==Limits of detection mini-lab==<br />
* [[Lab Manual: Limits of Detection]]<br />
* [[Lab Manual: Atomic Force Microscopy (AFM)]]<br />
* [[Limits of Detection: Data Sessions]]<br />
<br />
==MRI mini-lab==<br />
* [https://gate.nmr.mgh.harvard.edu/wiki/Tabletop_MRI/index.php/Main_Page MGH tabletop MRI]<br />
(Kept for historical reasons.)</div>Juliesuttonhttp://measurebiology.org/wiki/Spring_2020_Assignment_10Spring 2020 Assignment 102020-05-05T14:49:58Z<p>Juliesutton: </p>
<hr />
<div>In this assignment, you will find the amplitude and phase response of Hog1 to osmotic shock using student data from last semester. Note that this assignment is OPTIONAL, and completing it will give you +2 points of extra credit on a previous assignment. <br />
<br />
# Start by loading the data structure called <tt>fall2019_StudentData_Responses.mat</tt> into matlab. This file contains two variables: <br />
#* <tt>allClassResponseData</tt> is a cell array containing the analyzed response data from each group. Each element of the cell array is a 3-column matrix containing the response data. The columns represent (in order): the time (in seconds), the mean response (the average correlation between GFP and RFP in each cell), and the standard error of the response. To access the matrix of the <tt>ii</tt>th group, you can use curly braces: <tt>allClassResponseData{ii}</tt>.<br />
#* <tt>allClassOscillationPeriods</tt> is a vector containing the oscillation period (in seconds) for each of the elements in <tt>allClassResponseData</tt>.<br />
# For each set of data, use <tt>nlinfit</tt> to fit a sine function to extract the amplitude and phase of the response at that input frequency.<br />
#* Remember that <tt>nlinfit</tt> requires four inputs in this order: the independent variable (time), the dependent variable (hog1 response), a model function (more about this below), and a vector containing initial guesses for each parameter.<br />
#* Your model function should be a sine wave with an amplitude, phase, and offset (since the response data is not centered around zero) as fit parameters. The frequency of oscillations that we are interested in is determined by our experiment (given by <tt>1./allClassOscillationPeriods</tt>), and should not be left as a fit parameter.<br />
# Plot the best-fit amplitude and phase in a Bode plot.<br />
<br />
''Tip #1:'' Test your code for a single data set before looping through the entire cell array.<br />
<br />
''Tip #2:'' The model function used by <tt>nlinfit</tt> needs to have the vector of parameters as its first argument, and the independent variable as its second argument. You may find the following template useful for the model function:<br />
<br />
<pre><br />
exampleModelFunction = @(p,t) p(1) * t.^2 + p(2) * t + p(3) t^3;<br />
bestFitParameters = nlinfit( time, response, exampleModelFunction, [1 2 3]);<br />
</pre><br />
<br />
This is an example of the model function <math>y = a t^2 + b t +c</math>, where ''a, b,'' and ''c'' are fit parameters with initial guesses a = 1, b = 2, and c =3.<br />
<br />
{{Template:Assignment Turn In|message = <br />
<br />
# Write a function to fit the Hog1 response to a sinusoid and extract the estimated amplitude and phase shift of the response signal. For each set of response data, make a plot showing the Hog1 response vs time and the best fit model function. <br />
# Make a Bode plot (i.e. plot the amplitude and phase) of the Hog1-response as a function of frequency. Use log-log axes for the magnitude, and semi-log x axes for the phase. Since some frequencies have several measurements, you may either plot each fit result as a single point on the plot, or average the results together. For the phase plot, use <tt>wrapTo180</tt> or <tt>wrapToPi</tt> to make sure your phase is in the appropriate range of angles. <br />
# Qualitatively compare your Bode plot to the amplitude and phase found in the Mettetal paper. What aspects of the results are expected or unexpected? If your measurements do not agree, describe two or three reasons why they might differ.<br />
}}<br />
<br />
{{Template:Assignment Turn In|message = Turn in all your MATLAB code in pdf format. No need to include functions that you used but did not modify.}}</div>Juliesuttonhttp://measurebiology.org/wiki/Spring_2020_Assignment_10Spring 2020 Assignment 102020-05-04T21:24:22Z<p>Juliesutton: Created page with "# Start by loading the data structure called <tt>fall2019_StudentData_Responses.mat</tt> into matlab. This file contains two variables: #* <tt>allClassResponseData</tt> is a ..."</p>
<hr />
<div># Start by loading the data structure called <tt>fall2019_StudentData_Responses.mat</tt> into matlab. This file contains two variables: <br />
#* <tt>allClassResponseData</tt> is a cell array containing the analyzed response data from each group. Each element of the cell array is a 3-column matrix containing the response data. The columns represent (in order): the time (in seconds), the mean response (the average correlation between GFP and RFP in each cell), and the standard error of the response. To access the matrix of the <tt>ii</tt>th group, you can use curly braces: <tt>allClassResponseData{ii}</tt>.<br />
#* <tt>allClassOscillationPeriods</tt> is a vector containing the oscillation period (in seconds) for each of the elements in <tt>allClassResponseData</tt>.<br />
# For each set of data, fit a sine function to extract the amplitude and phase of the response at that input frequency.<br />
#* There are a few important, but subtle points here. <br />
#** Since the response data is not centered around zero, you must include a constant offset in your model function. <br />
#** The frequency of oscillations that we are interested in is determined by our experiment, and should not be left as a fit parameter. <br />
<br />
<br />
<br />
{{Template:Assignment Turn In|message = <br />
<br />
For each set of response data:<br />
# Plot the response vs time in one subplot, and the pinch valve state (1 for high salt, 0 for low salt) in a second subplot. <br />
# Write a function to fit the response to a sinusoid and extract the predicted amplitude and phase shift of the response signal. Plot the best fit function on the same plot as your data. <br />
# On one set of axes, plot the Hog1-response vs. time and the best fit sinusoid.<br />
# Make a Bode Plot (amplitude and phase) of the Hog1-response as a function of frequency.<br />
# How does your data compare to the amplitude and phase found in the paper? If your measurements do not agree, describe two or three reasons why they might differ.<br />
}}<br />
<br />
{{Template:Assignment Turn In|message = Turn in all your MATLAB code in pdf format. No need to include functions that you used but did not modify.}}</div>Juliesuttonhttp://measurebiology.org/wiki/20.309_Main_Page20.309 Main Page2020-05-04T21:07:32Z<p>Juliesutton: /* Module 2: electronics, signals and systems */</p>
<hr />
<div>{{Template:20.309}}<br />
__NOTOC__<br />
Bioinstrumentation scholars: you are welcome to edit and improve this course wiki. Correct, helpful, exquisitely-expressed changes will increase your class participation grade. Alterations not meeting those critera may have the opposite effect.<br />
<br />
If you would like to contribute to this site, please [[Special:RequestAccount|request an account]].<br />
<br />
== 20.309 course information==<br />
<br />
* [[20.309:Course Information| 20.309 course information]]<br />
* [[20.309:Safety| lab safety]]<br />
* Reserve a lab station here: [http://measurebiology.org/reservations/Web/schedule.php Bioinstrumentation Lab reservation system]<br />
<br />
== 20.309 master schedule==<br />
<br />
<div style="padding: 10px; width: 740px; border: 5px solid #000FFF;"><br />
===Module 1: Optics & Microscopy===<br />
{| border=1px<br />
|style="width: 10%;"|'''Important dates''' <br />
|style="width: 15%;"|'''Assignment due'''<br />
|style="width: 30%;"|'''Assignment page links'''<br />
|style="width: 45%;"|'''Useful resources'''<br />
|-- <br />
| <font color=#04B45F>2/16/20</font color><br />
| Assignment 1<br />
| <br />
[[Assignment 1 Overview: Transillumination microscopy| Assignment 1 Overview]]<br />
* [[Assignment 1, Part 1: Pre-lab questions|Part 1: Pre-lab questions]]<br />
* [[Assignment 1, Part 2: Optics bootcamp|Part 2: Optics bootcamp]]<br />
* [[Assignment 1, Part 3: Building your transillumination microscope|Part 3: Build a microscope]]<br />
* [[Assignment 1, Part 4: Building your transilluminated microscope|Part 4: Measure stuff]]<br />
| <br />
* [https://stellar.mit.edu/S/course/20/sp20/20.309/materials.html Lectures 1 through 9 of the 20.309 class]<br />
* [http://www.microscopyu.com/articles/formulas/formulasri.html Snell's law]<br />
* [[Understanding log plots]]<br />
|-<br />
| <font color=#04B45F>2/23/20</font color><br />
| Assignment 2<br />
| [[Assignment 2: fluorescence microscopy| Assignment 2 Overview]]<br />
* [[Assignment 2 Part 1: Noise in images|Part 1: Noise in images]]<br />
* [[Assignment 2 Part 2: Fluorescence microscopy|Part 2: Fluorescence microscopy]]<br />
* [[Assignment 2 Part 3: Build an epi-illuminator for your microscope|Part 3: Build an epi-illuminator for your microscope]]<br />
|<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [[Optical aberrations]]<br />
* [[Aperture and field stops]]<br />
* [[Optical detectors, noise, and the limit of detection]]<br />
* [[Manta G032 camera measurements]]<br />
|-<br />
| <font color=#04B45F>2/25/20</font color><br />
| Quiz 1 (in lecture)<br />
| ||<br />
|-<br />
| <font color=#04B45F>3/1/20</font color><br />
| Assignment 3<br />
| [[Assignment 3 Overview|Assignment 3 Overview]]<br />
* [[Assignment 3, Part 1: visualizing actin with fluorescence contrast|Part 1: fluorescence imaging]] <br />
* [[Assignment 3, Part 2: experimental design with fluorescence| Part 2: choosing filters]] <br />
|<br />
*[[Flat-field correction|Flat-field correction of non-uniform illumination]]<br />
*[[Matlab: Scalebars]]<br />
|-<br />
| <font color=#04B45F>3/8/20</font color><br />
| Assignment 4<br />
|{{Template:Assignment 4 navigation}}<br />
|<br />
* [http://www.microscopyu.com/articles/formulas/formulasresolution.html Resolution]<br />
* [[Physical optics and resolution]]<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[MATLAB: Estimating resolution from a PSF slide image]]<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
|-<br />
| <font color=#04B45F>4/3/20</font color><br />
| Assignment 5<br />
| [[Assignment 5: Spring 2020|Assignment 5 Overview (Spring 2020)]]<br />
* [[Assignment 5, Part 1: MSD difference tracking and microscope stability|Part 1: MSD difference tracking and microscope stability]] <br />
* [[Assignment 5, Part 2: live cell particle tracking of endocytosed beads| Part 2: Live cell particle tracking of endocytosed beads]]<br />
|<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
|-<br />
| <font color=#04B45F>4/7/20</font color><br />
| Exam 1 <br />
| <br />
|<br />
|-<br />
|}<br />
<br /><br />
<br />
===Module 2: electronics, signals and systems===<br />
{| border=1px<br />
|style="width: 10%;"|'''Important dates''' <br />
|style="width: 15%;"|'''Assignment due'''<br />
|style="width: 30%;"|'''Assignment page links'''<br />
|style="width: 45%;"|'''Useful resources'''<br />
|-- <br />
| <font color=#04B45F>4/13/20</font color><br />
| Assignment 6<br />
| [[Spring 2020 Assignment 6]]<br />
* (Previous semesters' assignment 6 for reference)<br />
* [[Assignment 6 Overview: two color microscope|Assignment 6 overview]]<br />
* [[Assignment 6, Part 1: build a two-color microscope]]<br />
* [[Assignment 6, Part 2: electronics written problems]]<br />
|<br />
* [[Electronics primer]]<br />
* [https://www.khanacademy.org/science/electrical-engineering/ee-circuit-analysis-topic Khan Academy Material, esp. "Ideal Circuit Elements" and "Voltage Divider"]<br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Input and output impedance]]<br />
|-<br />
| <font color=#04B45F>4/20/20</font color><br />
| Assignment 7<br />
| [[Spring 2020 Assignment 7]]<br />
* (Previous semesters' assignment 7 for reference)<br />
* [[Electronics boot camp I: passive circuits and transfer functions|Assignment 7 overview]]<br />
* [[Electronics written problems II|Assignment 7, Part 1: more electronics written problems]]<br />
* [[Electronics boot camp lab part 1|Assignment 7, Part 2: Electronics bootcamp I]]<br />
<br />
| <br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Bode plots|Transfer functions and Bode plots]]<br />
* [[Complex Number Review|Complex number review]]<br />
* [[Input and output impedance]]<br />
|-<br />
| <font color=#04B45F>4/21/20</font color><br />
| Quiz 2 (60 min)<br />
| <br />
|<br />
|-<br />
| <font color=#04B45F>4/27/20</font color><br />
| Assignment 8<br />
| [[Spring 2020 Assignment 8]]<br />
*(Previous semester's assignment 8 for reference)<br />
*[[Assignment 8 Overview: flow channel & two-color microscope|Assignment 8 Overview]]<br />
* [[Electronics bootcamp II: feedback systems|Part 1: feedback systems]]<br />
* [[Assignment 8, Part 2: fabricate a microfluidic device| Part 2: fabricate a microfluidic device]]<br />
* [[Assignment 8, Part 3: add flow control and test your device| Part 3: add flow control and test your device]]<br />
|[http://science.sciencemag.org/content/319/5862/482 *The* yeast paper: Mettelal ''et al.'', ''Science'' (2008)]<br />
|-<br />
| <font color=#04B45F>5/4/20</font color><br />
| Assignment 9<br />
| [[Spring 2020 Assignment 9]]<br />
*(Previous semester's assignment 9 for reference)<br />
* [[Assignment 9 Overview: Analyzing yeast images]]<br />
* [[Assignment 8, Part 0: convolution practice| Part 0: convolution practice]]<br />
* [[Assignment 9, Part 1: Analyze two-color yeast images| Part 1: Analyze two-color yeast images]]<br />
* [[Assignment 10, Part 1: Measuring the osmotic shock response of yeast| A10: Measure the osmotic shock response of yeast]]<br />
|<br />
|-<br />
| <font color=#04B45F>5/8/20</font color><br />
| Exam 2 (90 min)<br />
| <br />
|<br />
|-<br />
| <font color=#04B45F>5/12/20</font color><br />
| Assignment 10 <br />
| Spring 2020 Assignment 10<br />
*(Previous semester's assignment 10 was combined with assignment 9)<br />
|<br />
|-<br />
| cancelled<br />
| Assignment 11<br />
| [[Assignment 11: Tell us about your lab visit]]<br />
| <br />
|-<br />
|}<br />
</div><br />
<br />
==Optics and microscopy resources==<br />
===Background reading===<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [[Optical aberrations]]<br />
* [[Aperture and field stops]]<br />
* [[Optical detectors, noise, and the limit of detection]]<br />
* [[Manta G032 camera measurements]]<br />
* [[Understanding log plots]]<br />
<br />
===Code examples and simulations===<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[MATLAB: Estimating resolution from a PSF slide image]]<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
<br />
==Electronics resources==<br />
===Background reading===<br />
* [[Electronics primer]]<br />
* [https://www.khanacademy.org/science/electrical-engineering/ee-circuit-analysis-topic Khan Academy Material, esp. "Ideal Circuit Elements" and "Voltage Divider"]<br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Bode plots|Transfer functions and Bode plots]]<br />
* [[Complex Number Review|Complex number review]]<br />
* [[Input and output impedance]]<br />
<br />
==Limits of detection mini-lab==<br />
* [[Lab Manual: Limits of Detection]]<br />
* [[Lab Manual: Atomic Force Microscopy (AFM)]]<br />
* [[Limits of Detection: Data Sessions]]<br />
<br />
==MRI mini-lab==<br />
* [https://gate.nmr.mgh.harvard.edu/wiki/Tabletop_MRI/index.php/Main_Page MGH tabletop MRI]<br />
(Kept for historical reasons.)</div>Juliesuttonhttp://measurebiology.org/wiki/20.309_Main_Page20.309 Main Page2020-05-04T18:18:39Z<p>Juliesutton: /* Module 2: electronics, signals and systems */</p>
<hr />
<div>{{Template:20.309}}<br />
__NOTOC__<br />
Bioinstrumentation scholars: you are welcome to edit and improve this course wiki. Correct, helpful, exquisitely-expressed changes will increase your class participation grade. Alterations not meeting those critera may have the opposite effect.<br />
<br />
If you would like to contribute to this site, please [[Special:RequestAccount|request an account]].<br />
<br />
== 20.309 course information==<br />
<br />
* [[20.309:Course Information| 20.309 course information]]<br />
* [[20.309:Safety| lab safety]]<br />
* Reserve a lab station here: [http://measurebiology.org/reservations/Web/schedule.php Bioinstrumentation Lab reservation system]<br />
<br />
== 20.309 master schedule==<br />
<br />
<div style="padding: 10px; width: 740px; border: 5px solid #000FFF;"><br />
===Module 1: Optics & Microscopy===<br />
{| border=1px<br />
|style="width: 10%;"|'''Important dates''' <br />
|style="width: 15%;"|'''Assignment due'''<br />
|style="width: 30%;"|'''Assignment page links'''<br />
|style="width: 45%;"|'''Useful resources'''<br />
|-- <br />
| <font color=#04B45F>2/16/20</font color><br />
| Assignment 1<br />
| <br />
[[Assignment 1 Overview: Transillumination microscopy| Assignment 1 Overview]]<br />
* [[Assignment 1, Part 1: Pre-lab questions|Part 1: Pre-lab questions]]<br />
* [[Assignment 1, Part 2: Optics bootcamp|Part 2: Optics bootcamp]]<br />
* [[Assignment 1, Part 3: Building your transillumination microscope|Part 3: Build a microscope]]<br />
* [[Assignment 1, Part 4: Building your transilluminated microscope|Part 4: Measure stuff]]<br />
| <br />
* [https://stellar.mit.edu/S/course/20/sp20/20.309/materials.html Lectures 1 through 9 of the 20.309 class]<br />
* [http://www.microscopyu.com/articles/formulas/formulasri.html Snell's law]<br />
* [[Understanding log plots]]<br />
|-<br />
| <font color=#04B45F>2/23/20</font color><br />
| Assignment 2<br />
| [[Assignment 2: fluorescence microscopy| Assignment 2 Overview]]<br />
* [[Assignment 2 Part 1: Noise in images|Part 1: Noise in images]]<br />
* [[Assignment 2 Part 2: Fluorescence microscopy|Part 2: Fluorescence microscopy]]<br />
* [[Assignment 2 Part 3: Build an epi-illuminator for your microscope|Part 3: Build an epi-illuminator for your microscope]]<br />
|<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [[Optical aberrations]]<br />
* [[Aperture and field stops]]<br />
* [[Optical detectors, noise, and the limit of detection]]<br />
* [[Manta G032 camera measurements]]<br />
|-<br />
| <font color=#04B45F>2/25/20</font color><br />
| Quiz 1 (in lecture)<br />
| ||<br />
|-<br />
| <font color=#04B45F>3/1/20</font color><br />
| Assignment 3<br />
| [[Assignment 3 Overview|Assignment 3 Overview]]<br />
* [[Assignment 3, Part 1: visualizing actin with fluorescence contrast|Part 1: fluorescence imaging]] <br />
* [[Assignment 3, Part 2: experimental design with fluorescence| Part 2: choosing filters]] <br />
|<br />
*[[Flat-field correction|Flat-field correction of non-uniform illumination]]<br />
*[[Matlab: Scalebars]]<br />
|-<br />
| <font color=#04B45F>3/8/20</font color><br />
| Assignment 4<br />
|{{Template:Assignment 4 navigation}}<br />
|<br />
* [http://www.microscopyu.com/articles/formulas/formulasresolution.html Resolution]<br />
* [[Physical optics and resolution]]<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[MATLAB: Estimating resolution from a PSF slide image]]<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
|-<br />
| <font color=#04B45F>4/3/20</font color><br />
| Assignment 5<br />
| [[Assignment 5: Spring 2020|Assignment 5 Overview (Spring 2020)]]<br />
* [[Assignment 5, Part 1: MSD difference tracking and microscope stability|Part 1: MSD difference tracking and microscope stability]] <br />
* [[Assignment 5, Part 2: live cell particle tracking of endocytosed beads| Part 2: Live cell particle tracking of endocytosed beads]]<br />
|<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
|-<br />
| <font color=#04B45F>4/7/20</font color><br />
| Exam 1 <br />
| <br />
|<br />
|-<br />
|}<br />
<br /><br />
<br />
===Module 2: electronics, signals and systems===<br />
{| border=1px<br />
|style="width: 10%;"|'''Important dates''' <br />
|style="width: 15%;"|'''Assignment due'''<br />
|style="width: 30%;"|'''Assignment page links'''<br />
|style="width: 45%;"|'''Useful resources'''<br />
|-- <br />
| <font color=#04B45F>4/13/20</font color><br />
| Assignment 6<br />
| [[Spring 2020 Assignment 6]]<br />
* (Previous semesters' assignment 6 for reference)<br />
* [[Assignment 6 Overview: two color microscope|Assignment 6 overview]]<br />
* [[Assignment 6, Part 1: build a two-color microscope]]<br />
* [[Assignment 6, Part 2: electronics written problems]]<br />
|<br />
* [[Electronics primer]]<br />
* [https://www.khanacademy.org/science/electrical-engineering/ee-circuit-analysis-topic Khan Academy Material, esp. "Ideal Circuit Elements" and "Voltage Divider"]<br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Input and output impedance]]<br />
|-<br />
| <font color=#04B45F>4/20/20</font color><br />
| Assignment 7<br />
| [[Spring 2020 Assignment 7]]<br />
* (Previous semesters' assignment 7 for reference)<br />
* [[Electronics boot camp I: passive circuits and transfer functions|Assignment 7 overview]]<br />
* [[Electronics written problems II|Assignment 7, Part 1: more electronics written problems]]<br />
* [[Electronics boot camp lab part 1|Assignment 7, Part 2: Electronics bootcamp I]]<br />
<br />
| <br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Bode plots|Transfer functions and Bode plots]]<br />
* [[Complex Number Review|Complex number review]]<br />
* [[Input and output impedance]]<br />
|-<br />
| <font color=#04B45F>4/21/20</font color><br />
| Quiz 2 (60 min)<br />
| <br />
|<br />
|-<br />
| <font color=#04B45F>4/27/20</font color><br />
| Assignment 8<br />
| [[Spring 2020 Assignment 8]]<br />
*(Previous semester's assignment 8 for reference)<br />
*[[Assignment 8 Overview: flow channel & two-color microscope|Assignment 8 Overview]]<br />
* [[Electronics bootcamp II: feedback systems|Part 1: feedback systems]]<br />
* [[Assignment 8, Part 2: fabricate a microfluidic device| Part 2: fabricate a microfluidic device]]<br />
* [[Assignment 8, Part 3: add flow control and test your device| Part 3: add flow control and test your device]]<br />
|[http://science.sciencemag.org/content/319/5862/482 *The* yeast paper: Mettelal ''et al.'', ''Science'' (2008)]<br />
|-<br />
| <font color=#04B45F>5/4/20</font color><br />
| Assignment 9<br />
| [[Spring 2020 Assignment 9]]<br />
*(Previous semester's assignment 9 for reference)<br />
* [[Assignment 9 Overview: Analyzing yeast images]]<br />
* [[Assignment 8, Part 0: convolution practice| Part 0: convolution practice]]<br />
* [[Assignment 9, Part 1: Analyze two-color yeast images| Part 1: Analyze two-color yeast images]]<br />
* [[Assignment 10, Part 1: Measuring the osmotic shock response of yeast| A10: Measure the osmotic shock response of yeast]]<br />
|<br />
|-<br />
| <font color=#04B45F>5/8/20</font color><br />
| Exam 2 (90 min)<br />
| <br />
|<br />
|-<br />
| <font color=#04B45F>5/12/20</font color><br />
| Assignment 10 <br />
| [[Spring 2020 Assignment 10]]<br />
*(Previous semester's assignment 10 was combined with assignment 9)<br />
|<br />
|-<br />
| cancelled<br />
| Assignment 11<br />
| [[Assignment 11: Tell us about your lab visit]]<br />
| <br />
|-<br />
|}<br />
</div><br />
<br />
==Optics and microscopy resources==<br />
===Background reading===<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [[Optical aberrations]]<br />
* [[Aperture and field stops]]<br />
* [[Optical detectors, noise, and the limit of detection]]<br />
* [[Manta G032 camera measurements]]<br />
* [[Understanding log plots]]<br />
<br />
===Code examples and simulations===<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[MATLAB: Estimating resolution from a PSF slide image]]<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
<br />
==Electronics resources==<br />
===Background reading===<br />
* [[Electronics primer]]<br />
* [https://www.khanacademy.org/science/electrical-engineering/ee-circuit-analysis-topic Khan Academy Material, esp. "Ideal Circuit Elements" and "Voltage Divider"]<br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Bode plots|Transfer functions and Bode plots]]<br />
* [[Complex Number Review|Complex number review]]<br />
* [[Input and output impedance]]<br />
<br />
==Limits of detection mini-lab==<br />
* [[Lab Manual: Limits of Detection]]<br />
* [[Lab Manual: Atomic Force Microscopy (AFM)]]<br />
* [[Limits of Detection: Data Sessions]]<br />
<br />
==MRI mini-lab==<br />
* [https://gate.nmr.mgh.harvard.edu/wiki/Tabletop_MRI/index.php/Main_Page MGH tabletop MRI]<br />
(Kept for historical reasons.)</div>Juliesuttonhttp://measurebiology.org/wiki/20.309_Main_Page20.309 Main Page2020-04-30T13:20:34Z<p>Juliesutton: /* Module 2: electronics, signals and systems */</p>
<hr />
<div>{{Template:20.309}}<br />
__NOTOC__<br />
Bioinstrumentation scholars: you are welcome to edit and improve this course wiki. Correct, helpful, exquisitely-expressed changes will increase your class participation grade. Alterations not meeting those critera may have the opposite effect.<br />
<br />
If you would like to contribute to this site, please [[Special:RequestAccount|request an account]].<br />
<br />
== 20.309 course information==<br />
<br />
* [[20.309:Course Information| 20.309 course information]]<br />
* [[20.309:Safety| lab safety]]<br />
* Reserve a lab station here: [http://measurebiology.org/reservations/Web/schedule.php Bioinstrumentation Lab reservation system]<br />
<br />
== 20.309 master schedule==<br />
<br />
<div style="padding: 10px; width: 740px; border: 5px solid #000FFF;"><br />
===Module 1: Optics & Microscopy===<br />
{| border=1px<br />
|style="width: 10%;"|'''Important dates''' <br />
|style="width: 15%;"|'''Assignment due'''<br />
|style="width: 30%;"|'''Assignment page links'''<br />
|style="width: 45%;"|'''Useful resources'''<br />
|-- <br />
| <font color=#04B45F>2/16/20</font color><br />
| Assignment 1<br />
| <br />
[[Assignment 1 Overview: Transillumination microscopy| Assignment 1 Overview]]<br />
* [[Assignment 1, Part 1: Pre-lab questions|Part 1: Pre-lab questions]]<br />
* [[Assignment 1, Part 2: Optics bootcamp|Part 2: Optics bootcamp]]<br />
* [[Assignment 1, Part 3: Building your transillumination microscope|Part 3: Build a microscope]]<br />
* [[Assignment 1, Part 4: Building your transilluminated microscope|Part 4: Measure stuff]]<br />
| <br />
* [https://stellar.mit.edu/S/course/20/sp20/20.309/materials.html Lectures 1 through 9 of the 20.309 class]<br />
* [http://www.microscopyu.com/articles/formulas/formulasri.html Snell's law]<br />
* [[Understanding log plots]]<br />
|-<br />
| <font color=#04B45F>2/23/20</font color><br />
| Assignment 2<br />
| [[Assignment 2: fluorescence microscopy| Assignment 2 Overview]]<br />
* [[Assignment 2 Part 1: Noise in images|Part 1: Noise in images]]<br />
* [[Assignment 2 Part 2: Fluorescence microscopy|Part 2: Fluorescence microscopy]]<br />
* [[Assignment 2 Part 3: Build an epi-illuminator for your microscope|Part 3: Build an epi-illuminator for your microscope]]<br />
|<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [[Optical aberrations]]<br />
* [[Aperture and field stops]]<br />
* [[Optical detectors, noise, and the limit of detection]]<br />
* [[Manta G032 camera measurements]]<br />
|-<br />
| <font color=#04B45F>2/25/20</font color><br />
| Quiz 1 (in lecture)<br />
| ||<br />
|-<br />
| <font color=#04B45F>3/1/20</font color><br />
| Assignment 3<br />
| [[Assignment 3 Overview|Assignment 3 Overview]]<br />
* [[Assignment 3, Part 1: visualizing actin with fluorescence contrast|Part 1: fluorescence imaging]] <br />
* [[Assignment 3, Part 2: experimental design with fluorescence| Part 2: choosing filters]] <br />
|<br />
*[[Flat-field correction|Flat-field correction of non-uniform illumination]]<br />
*[[Matlab: Scalebars]]<br />
|-<br />
| <font color=#04B45F>3/8/20</font color><br />
| Assignment 4<br />
|{{Template:Assignment 4 navigation}}<br />
|<br />
* [http://www.microscopyu.com/articles/formulas/formulasresolution.html Resolution]<br />
* [[Physical optics and resolution]]<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[MATLAB: Estimating resolution from a PSF slide image]]<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
|-<br />
| <font color=#04B45F>4/3/20</font color><br />
| Assignment 5<br />
| [[Assignment 5: Spring 2020|Assignment 5 Overview (Spring 2020)]]<br />
* [[Assignment 5, Part 1: MSD difference tracking and microscope stability|Part 1: MSD difference tracking and microscope stability]] <br />
* [[Assignment 5, Part 2: live cell particle tracking of endocytosed beads| Part 2: Live cell particle tracking of endocytosed beads]]<br />
|<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
|-<br />
| <font color=#04B45F>4/7/20</font color><br />
| Exam 1 <br />
| <br />
|<br />
|-<br />
|}<br />
<br /><br />
<br />
===Module 2: electronics, signals and systems===<br />
{| border=1px<br />
|style="width: 10%;"|'''Important dates''' <br />
|style="width: 15%;"|'''Assignment due'''<br />
|style="width: 30%;"|'''Assignment page links'''<br />
|style="width: 45%;"|'''Useful resources'''<br />
|-- <br />
| <font color=#04B45F>4/13/20</font color><br />
| Assignment 6<br />
| [[Spring 2020 Assignment 6]]<br />
* (Previous semesters' assignment 6 for reference)<br />
* [[Assignment 6 Overview: two color microscope|Assignment 6 overview]]<br />
* [[Assignment 6, Part 1: build a two-color microscope]]<br />
* [[Assignment 6, Part 2: electronics written problems]]<br />
|<br />
* [[Electronics primer]]<br />
* [https://www.khanacademy.org/science/electrical-engineering/ee-circuit-analysis-topic Khan Academy Material, esp. "Ideal Circuit Elements" and "Voltage Divider"]<br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Input and output impedance]]<br />
|-<br />
| <font color=#04B45F>4/20/20</font color><br />
| Assignment 7<br />
| [[Spring 2020 Assignment 7]]<br />
* (Previous semesters' assignment 7 for reference)<br />
* [[Electronics boot camp I: passive circuits and transfer functions|Assignment 7 overview]]<br />
* [[Electronics written problems II|Assignment 7, Part 1: more electronics written problems]]<br />
* [[Electronics boot camp lab part 1|Assignment 7, Part 2: Electronics bootcamp I]]<br />
<br />
| <br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Bode plots|Transfer functions and Bode plots]]<br />
* [[Complex Number Review|Complex number review]]<br />
* [[Input and output impedance]]<br />
|-<br />
| <font color=#04B45F>4/21/20</font color><br />
| Quiz 2 (60 min)<br />
| <br />
|<br />
|-<br />
| <font color=#04B45F>4/27/20</font color><br />
| Assignment 8<br />
| [[Spring 2020 Assignment 8]]<br />
*(Previous semester's assignment 8 for reference)<br />
*[[Assignment 8 Overview: flow channel & two-color microscope|Assignment 8 Overview]]<br />
* [[Electronics bootcamp II: feedback systems|Part 1: feedback systems]]<br />
* [[Assignment 8, Part 2: fabricate a microfluidic device| Part 2: fabricate a microfluidic device]]<br />
* [[Assignment 8, Part 3: add flow control and test your device| Part 3: add flow control and test your device]]<br />
|[http://science.sciencemag.org/content/319/5862/482 *The* yeast paper: Mettelal ''et al.'', ''Science'' (2008)]<br />
|-<br />
| <font color=#04B45F>5/4/20</font color><br />
| Assignment 9<br />
| [[Spring 2020 Assignment 9]]<br />
*(Previous semester's assignment 9 for reference)<br />
* [[Assignment 9 Overview: Analyzing yeast images]]<br />
* [[Assignment 8, Part 0: convolution practice| Part 0: convolution practice]]<br />
* [[Assignment 9, Part 1: Analyze two-color yeast images| Part 1: Analyze two-color yeast images]]<br />
* [[Assignment 10, Part 1: Measuring the osmotic shock response of yeast| A10: Measure the osmotic shock response of yeast]]<br />
|<br />
|-<br />
| <font color=#04B45F>5/12/20</font color><br />
| Assignment 10 <br />
| [[Spring 2020 Assignment 10]]<br />
*(Previous semester's assignment 10 was combined with assignment 9)<br />
|<br />
|-<br />
| <font color=#04B45F>5/7/20</font color><br />
| Exam 2 (90 min)<br />
| <br />
|<br />
|-<br />
| cancelled<br />
| Assignment 11<br />
| [[Assignment 11: Tell us about your lab visit]]<br />
| <br />
|-<br />
|}<br />
</div><br />
<br />
==Optics and microscopy resources==<br />
===Background reading===<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [[Optical aberrations]]<br />
* [[Aperture and field stops]]<br />
* [[Optical detectors, noise, and the limit of detection]]<br />
* [[Manta G032 camera measurements]]<br />
* [[Understanding log plots]]<br />
<br />
===Code examples and simulations===<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[MATLAB: Estimating resolution from a PSF slide image]]<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
<br />
==Electronics resources==<br />
===Background reading===<br />
* [[Electronics primer]]<br />
* [https://www.khanacademy.org/science/electrical-engineering/ee-circuit-analysis-topic Khan Academy Material, esp. "Ideal Circuit Elements" and "Voltage Divider"]<br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Bode plots|Transfer functions and Bode plots]]<br />
* [[Complex Number Review|Complex number review]]<br />
* [[Input and output impedance]]<br />
<br />
==Limits of detection mini-lab==<br />
* [[Lab Manual: Limits of Detection]]<br />
* [[Lab Manual: Atomic Force Microscopy (AFM)]]<br />
* [[Limits of Detection: Data Sessions]]<br />
<br />
==MRI mini-lab==<br />
* [https://gate.nmr.mgh.harvard.edu/wiki/Tabletop_MRI/index.php/Main_Page MGH tabletop MRI]<br />
(Kept for historical reasons.)</div>Juliesuttonhttp://measurebiology.org/wiki/Spring_2020_Assignment_9Spring 2020 Assignment 92020-04-28T18:33:58Z<p>Juliesutton: /* Yeast experiment data */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:Electronics]]<br />
{{Template:20.309}}<br />
__NOTOC__<br />
==Signals and systems==<br />
{{Template:Assignment Turn In|message =<br />
Fill out the table below. Match each system function with its Bode magnitude and phase plot, step response, and pole zero diagram. (Write one letter A-E in each box below.) In the row labeled “Description,” write a descriptive name of each system, such as “low-pass filter” or “overdamped second-order system.”<br />
}}<br />
<br />
{| border="1" style="width: 85%;"<br />
!System function<br />
!<math>\frac{1}{s+1}</math><br />
!<math>\frac{s}{s+1}</math><br />
!<math>\frac{s}{s^2+2s+1}</math><br />
!<math>\frac{s}{s^2+0.1s+1}</math><br />
!<math>\frac{1}{s^2+10s+1}</math><br />
|-<br />
!Magnitude plot<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
!Phase plot<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
!Step response<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
!Pole/zero plot<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
!Description<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|}<br />
<center><br />
'''Magnitude Plots'''<br />
<br />
[[File:Transfer function matching magnitude plots.png|700 px]]<br />
<br />
'''Phase Plots'''<br />
<br />
[[File:Transfer function matching phase plots.png|700 px]]<br />
<br />
'''Step Response Plots'''<br />
<br />
[[File:Transfer function matching step response plots.png|700 px]]<br />
<br />
'''Pole Zero Plots'''<br />
<br />
[[File:Transfer function matching pole zero plots.png|700 px]]<br />
</center><br />
{{Template:Assignment Turn In|message =<br />
* Use graphical methods to find the Fourier transform of the half-cosine pulse function x(t) plotted below, which consists of the positive half of a 1 Hz cosine, repeated forever at a rate of 1 Hz.<br />
* What is the lowest frequency component of x(t), not counting <math>\hat{X}(0)</math>? <br />
}}<br />
<br />
[[File:Cosine pulse function.png|700 px]]<br />
One way to create x(t) using functions that appear on the transform table is:<br />
# '''multiply''' a '''cosine''' by a '''rectangle''', and then<br />
# '''convolve''' the result with the '''comb function''' <math>\mathrm{III(}t)=\sum\limits_{n=-∞}^{∞} \delta(t-nT)</math>. <br />
Use the diagram below to help you find the answer. The left column of shows signals in the time domain, and the right column shows the magnitude of the Fourier transform of each signal. The top right plot is filled in for you, plus a little hint that might help you make an accurate plot. <br />
<br />
(The phase of the transforms in this problem is zero at all frequencies, so it is not plotted.)<br />
<br />
[[File:Cosine pulse transform worksheet.png|700 px]]<br />
<br />
==Feedback systems==<br />
{{Template:Assignment Turn In|message =<br />
<ul><br />
<li>Find the transfer function H ̂(s)=(V ̂_out (s))/(V ̂_in (s)) of the circuit shown below, assuming L = 1 H and R = 1&Omega;.</li><br />
<li>Plot the poles and zeros of H ̂(s) on a set of axes using x’s for poles and o’s for zeroes.</li><br />
<li>The circuit from Figure 1 is placed in a feedback system, as shown in the block diagram below. The triangle represents an amplifier with gain G that does not depend on frequency. Find the transfer function of the feedback system <math>\hat{F}(s)=(\hat{Y}(s))/(\hat{X}(s)).</math></li><br />
<li>Plot the poles and zeros of <math>\hat{F}(s)</math> for G=1,9,and 19. Label the gain value for each point.</math><br />
</ul><br />
}}<br />
<br />
<center><br />
{|<br />
|[[File:LR Low Pass Filter for S20 Assignment 9.png|250 px]]<br />
|[[File:Feedback System Block Diagram for S20 Assignment 9.png|350 px]]<br />
|}<br />
</center><br />
<br />
{{Template:Assignment Turn In|message =<br />
# Find the transfer function of the circuit below for L = 1 H and R = 1 &Omega; and C=1 F.<br />
# The circuit is placed in the same feedback system shown in the previous question. Plot the poles for gains of 1/4, 3/4, 5/4, 10/4, and 17/4. You may generate your plot by hand or use MATLAB.<br />
}}<br />
<center>[[File:LRC circuit for SP20 assignment 9.png|250 px]]</center><br />
<br />
==The Frequency Dependence of Osmo-Adaptation in Saccharomyces cerevisiae==<br />
{{Template:Assignment Turn In|message =<br />
Read [https://www.dropbox.com/s/cmgq0b33vn8csow/Frequency%20Dependence%20of%20Osmo-Adaptation%20in%20Saccharomyces%20cerevisiae.pdf?dl=0 The Frequency Dependence of Osmo-Adaptation in Saccharomyces cerevisiae] and the [https://www.dropbox.com/s/y9k4qfzg8uld656/The%20Frequency%20Dependence%20of%20Osmo-Adaptation%20in%20Saccharomyces%20cerevisiae%20supporting.pdf?dl=0 supporting information.]. '''This paper will be the focus of exam 2.''' We will discuss the paper and the supporting information on Thursday and Friday (4/30 and 5/1).<br />
<br />
Answer the following questions about The Frequency Dependence of Osmo-Adaptation in S. cerevisiae:<br />
<ol><br />
<li>What is the primary mechanism by which S. cerevisiae recovers from hyperosmotic shock?</li><br />
<li>What mathematical model did Mettetal, ''et. al.'' use for Hog1 activation in response to a hyperosmotic shock? Express the model in the following forms<br />
<ul><br />
<li>transfer function (TF)</li><br />
<li>poles and zeros (ZPK)</li><br />
<li>single differential equation (SDE)</li><br />
<li>coupled differential equations (CDE)</li> <br />
</ul>Express the TF, SDE, and ZPK models in terms of the undamped natural frequency, <math>\omega_0</math>, damping ratio <math>\zeta</math>, and/or damped natural frequency <math>\omega_D</math>.</li><br />
<li>What mathematical model did Mettetal, et. al. use to account for nonlinearities in the system?</li><br />
<li>Plot the frequency response (i.e. make a Bode plot) of the model over a range of <math>\omega_0</math> and <math>\zeta</math> values that includes over damped, critically damped, and under damped.</li><br />
<li>Find an expression for the step response and plot it over a range of values of <math>\omega_0</math> and <math>\zeta</math>. A hand-drawn plot is fine, but you should probably look into MATLAB's <tt>step</tt> function.</li><br />
<li>Mettetal, et. al. found that the hyperosmotic shock response of wild-type yeast was (choose one): underdamped, critically damped, or overdamped.</li><br />
<li>The response of the mutant (low Pbs) yeast was (choose one): underdamped, critically damped, or overdamped.</li><br />
<li>Which of the step responses below corresponds to Mettetal's model for the wild-type strain and the mutant strain (neglecting the nonlinear element)?</li><br />
<li>Which of the Bode plots below corresponds to Mettetal's model for the wild-type strain and the mutant strain?</li><br />
<li>Which of the pole zero diagrams below corresponds to Mettetal's model for the wild-type strain and the mutant strain?</li><br />
<li>What are two questions that you have about the paper's methodology?</li><br />
}}<br />
<br />
<center><br />
{|<br />
|[[file:Mettetal yeast model step response.png|250 px]]<br />
|[[file:Mettetal yeast model Bode plots.png|250 px]]<br />
|[[file:Mettetal yeast model pole zero diagrams.png|250 px]]<br />
|}<br />
</center><br />
<br />
==Yeast experiment data==<br />
Unfortunately, we won't be collecting our own data in the lab this semester, but it's still important to get a feel for what the raw data looks like and how the signal manifests itself in those images. <br />
<br />
* Download the data file <tt>'fall2019_StudentData_3.mat'</tt> from the [https://www.dropbox.com/sh/j9gdbaoypukdydd/AACP1c3w6JzyzAa--XVpgMV0a?dl=0 Assignment 9 folder] in the course dropbox. The file contains raw data collected by 20.309 students during the Fall 2019 semester. <br />
* Load the file into your MATLAB workspace. You should see a variable called <tt>yeastOsmoticShockData</tt>, a struct which contains the movie data plus other relevant experimental parameters:<br />
<pre><br />
>> yeastOsmoticShockData<br />
<br />
yeastOsmoticShockData = <br />
<br />
struct with fields:<br />
<br />
Movie: [544×728×2×32 uint16]<br />
Time: [32×2 double]<br />
ValveState: [32×1 logical]<br />
ValveOscillationPeriod: 480<br />
BlueCameraGainAndExposure: [3 5000000]<br />
GreenCameraGainAndExposure: [15 5000000]<br />
</pre><br />
<br />
The engineered cells we used had 2 fluorescent proteins: a Hog1-GFP fusion and a nuclear protein tagged with RFP. Accordingly, the movie data has 2 color planes. The size of the movie's third dimension is 2. (It was 1 for the monochrome images we took in lab earlier this semester.) <br />
<br />
* Color plane 1 is the GFP-Hog1 image, taken with blue illumination and green emission. <br />
* The second color plane shows the RFP nuclear tag protein, taken with yellow/green illumination and red emission filters.<br />
<br />
Use <tt>implay</tt> to watch each color of the movie:<br />
<pre><br />
implay(double(yeastOsmoticShockData.Movie(:,:,1,:))/4095);<br />
implay(double(yeastOsmoticShockData.Movie(:,:,2,:))/4095);<br />
</pre><br />
<br />
Can you see how the distribution of Hog1 changes when the cell undergoes osmotic shock?<br />
<br />
Another way to visualize the two colors of this movie is to overlay them and view them as, well, a color movie. You can choose whatever color you want to display each signal. In the code below, the GFP signal is shown in red, and the RFP signal will be shown in cyan. Additionally, the code normalizes each frame to optimize the contrast, and adds a blue dot to the top left corner of the movie to indicate when the yeast culture medium has been switched to the 'high salt' state. <br />
<br />
<pre><br />
movie = zeros(size(yeastOsmoticShockData.Movie,1), size(yeastOsmoticShockData.Movie,2), 3, numberOfFrames);<br />
stateIndicator = createMaskFromCircles([50 50],10, [size(yeastOsmoticShockData.Movie,1), size(yeastOsmoticShockData.Movie,2)]);<br />
<br />
close all<br />
for ii = 1: size( yeastOsmoticShockData.Movie, 4 )<br />
normalize = @( x ) ( x - min( x(:) ) ) / range( x(:) );<br />
gfpImage = normalize( double( yeastOsmoticShockData.Movie(:,:,1,ii) ) );<br />
rfpImage = normalize( double( yeastOsmoticShockData.Movie(:,:,2,ii) ) );<br />
movie(:,:,:,ii) = ( cat( 3, gfpImage, rfpImage, rfpImage + stateIndicator*yeastOsmoticShockData.ValveState(ii) ) );<br />
end<br />
<br />
implay(movie)<br />
<br />
function Im = createMaskFromCircles(centers,radii,ImageSize)<br />
Im = zeros(ImageSize);<br />
[X, Y] = meshgrid(1:ImageSize(2), 1:1:ImageSize(1));<br />
<br />
for ii = 1:length(radii)<br />
isBright = (X-centers(ii,1)).^2+(Y-centers(ii,2)).^2 <= radii(ii,1)^2;<br />
isAlreadyFound = (Im == 1);<br />
Im = Im + (isBright & ~isAlreadyFound);<br />
end<br />
Im( Im > 1 ) = 1;<br />
<br />
end<br />
</pre><br />
<br />
{{Template:Assignment Turn In|message =<br />
# Step through the frames of the movie using the implay controls (for either the raw data or the color movie, your choice). Identify a frame where the signal is "high" - in other words, where the Hog1 signal is localized in the nucleus. Turn in the frame number that you've identified and a screen shot of the GFP-Hog1 movie at that frame number.<br />
# Repeat for a frame where the signal is "low" - in other words, where the Hog1 signal is uniformly distributed throughout the cell. Turn in the frame number that you've identified and a screen shot of the GFP-Hog1 movie at that frame number.<br />
}}<br />
<br />
{{Template:20.309 bottom}}</div>Juliesuttonhttp://measurebiology.org/wiki/Spring_2020_Assignment_9Spring 2020 Assignment 92020-04-28T14:25:44Z<p>Juliesutton: /* The Frequency Dependence of Osmo-Adaptation in Saccharomyces cerevisiae */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:Electronics]]<br />
{{Template:20.309}}<br />
__NOTOC__<br />
==Signals and systems==<br />
{{Template:Assignment Turn In|message =<br />
Fill out the table below. Match each system function with its Bode magnitude and phase plot, step response, and pole zero diagram. (Write one letter A-E in each box below.) In the row labeled “Description,” write a descriptive name of each system, such as “low-pass filter” or “overdamped second-order system.”<br />
}}<br />
<br />
{| border="1" style="width: 85%;"<br />
!System function<br />
!<math>\frac{1}{s+1}</math><br />
!<math>\frac{s}{s+1}</math><br />
!<math>\frac{s}{s^2+2s+1}</math><br />
!<math>\frac{s}{s^2+0.1s+1}</math><br />
!<math>\frac{1}{s^2+10s+1}</math><br />
|-<br />
!Magnitude plot<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
!Phase plot<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
!Step response<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
!Pole/zero plot<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
!Description<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|}<br />
<center><br />
'''Magnitude Plots'''<br />
<br />
[[File:Transfer function matching magnitude plots.png|700 px]]<br />
<br />
'''Phase Plots'''<br />
<br />
[[File:Transfer function matching phase plots.png|700 px]]<br />
<br />
'''Step Response Plots'''<br />
<br />
[[File:Transfer function matching step response plots.png|700 px]]<br />
<br />
'''Pole Zero Plots'''<br />
<br />
[[File:Transfer function matching pole zero plots.png|700 px]]<br />
</center><br />
{{Template:Assignment Turn In|message =<br />
* Use graphical methods to find the Fourier transform of the half-cosine pulse function x(t) plotted below, which consists of the positive half of a 1 Hz cosine, repeated forever at a rate of 1 Hz.<br />
* What is the lowest frequency component of x(t), not counting <math>\hat{X}(0)</math>? <br />
}}<br />
<br />
[[File:Cosine pulse function.png|700 px]]<br />
One way to create x(t) using functions that appear on the transform table is:<br />
# '''multiply''' a '''cosine''' by a '''rectangle''', and then<br />
# '''convolve''' the result with the '''comb function''' <math>\mathrm{III(}t)=\sum\limits_{n=-∞}^{∞} \delta(t-nT)</math>. <br />
Use the diagram below to help you find the answer. The left column of shows signals in the time domain, and the right column shows the magnitude of the Fourier transform of each signal. The top right plot is filled in for you, plus a little hint that might help you make an accurate plot. <br />
<br />
(The phase of the transforms in this problem is zero at all frequencies, so it is not plotted.)<br />
<br />
[[File:Cosine pulse transform worksheet.png|700 px]]<br />
<br />
==Feedback systems==<br />
{{Template:Assignment Turn In|message =<br />
<ul><br />
<li>Find the transfer function H ̂(s)=(V ̂_out (s))/(V ̂_in (s)) of the circuit shown below, assuming L = 1 H and R = 1&Omega;.</li><br />
<li>Plot the poles and zeros of H ̂(s) on a set of axes using x’s for poles and o’s for zeroes.</li><br />
<li>The circuit from Figure 1 is placed in a feedback system, as shown in the block diagram below. The triangle represents an amplifier with gain G that does not depend on frequency. Find the transfer function of the feedback system <math>\hat{F}(s)=(\hat{Y}(s))/(\hat{X}(s)).</math></li><br />
<li>Plot the poles and zeros of <math>\hat{F}(s)</math> for G=1,9,and 19. Label the gain value for each point.</math><br />
</ul><br />
}}<br />
<br />
<center><br />
{|<br />
|[[File:LR Low Pass Filter for S20 Assignment 9.png|250 px]]<br />
|[[File:Feedback System Block Diagram for S20 Assignment 9.png|350 px]]<br />
|}<br />
</center><br />
<br />
{{Template:Assignment Turn In|message =<br />
# Find the transfer function of the circuit below for L = 1 H and R = 1 &Omega; and C=1 F.<br />
# The circuit is placed in the same feedback system shown in the previous question. Plot the poles for gains of 1/4, 3/4, 5/4, 10/4, and 17/4. You may generate your plot by hand or use MATLAB.<br />
}}<br />
<center>[[File:LRC circuit for SP20 assignment 9.png|250 px]]</center><br />
<br />
==The Frequency Dependence of Osmo-Adaptation in Saccharomyces cerevisiae==<br />
{{Template:Assignment Turn In|message =<br />
Read [https://www.dropbox.com/s/cmgq0b33vn8csow/Frequency%20Dependence%20of%20Osmo-Adaptation%20in%20Saccharomyces%20cerevisiae.pdf?dl=0 The Frequency Dependence of Osmo-Adaptation in Saccharomyces cerevisiae] and the [https://www.dropbox.com/s/y9k4qfzg8uld656/The%20Frequency%20Dependence%20of%20Osmo-Adaptation%20in%20Saccharomyces%20cerevisiae%20supporting.pdf?dl=0 supporting information.]. '''This paper will be the focus of exam 2.''' We will discuss the paper and the supporting information on Thursday and Friday (4/30 and 5/1).<br />
<br />
Answer the following questions about The Frequency Dependence of Osmo-Adaptation in S. cerevisiae:<br />
<ol><br />
<li>What is the primary mechanism by which S. cerevisiae recovers from hyperosmotic shock?</li><br />
<li>What mathematical model did Mettetal, ''et. al.'' use for Hog1 activation in response to a hyperosmotic shock? Express the model in the following forms<br />
<ul><br />
<li>transfer function (TF)</li><br />
<li>poles and zeros (ZPK)</li><br />
<li>single differential equation (SDE)</li><br />
<li>coupled differential equations (CDE)</li> <br />
</ul>Express the TF, SDE, and ZPK models in terms of the undamped natural frequency, <math>\omega_0</math>, damping ratio <math>\zeta</math>, and/or damped natural frequency <math>\omega_D</math>.</li><br />
<li>What mathematical model did Mettetal, et. al. use to account for nonlinearities in the system?</li><br />
<li>Plot the frequency response (i.e. make a Bode plot) of the model over a range of <math>\omega_0</math> and <math>\zeta</math> values that includes over damped, critically damped, and under damped.</li><br />
<li>Find an expression for the step response and plot it over a range of values of <math>\omega_0</math> and <math>\zeta</math>. A hand-drawn plot is fine, but you should probably look into MATLAB's <tt>step</tt> function.</li><br />
<li>Mettetal, et. al. found that the hyperosmotic shock response of wild-type yeast was (choose one): underdamped, critically damped, or overdamped.</li><br />
<li>The response of the mutant (low Pbs) yeast was (choose one): underdamped, critically damped, or overdamped.</li><br />
<li>Which of the step responses below corresponds to Mettetal's model for the wild-type strain and the mutant strain (neglecting the nonlinear element)?</li><br />
<li>Which of the Bode plots below corresponds to Mettetal's model for the wild-type strain and the mutant strain?</li><br />
<li>Which of the pole zero diagrams below corresponds to Mettetal's model for the wild-type strain and the mutant strain?</li><br />
<li>What are two questions that you have about the paper's methodology?</li><br />
}}<br />
<br />
<center><br />
{|<br />
|[[file:Mettetal yeast model step response.png|250 px]]<br />
|[[file:Mettetal yeast model Bode plots.png|250 px]]<br />
|[[file:Mettetal yeast model pole zero diagrams.png|250 px]]<br />
|}<br />
</center><br />
<br />
Unfortunately, we won't be collecting our own data in the lab this semester, but it's still important to have a feel for what the raw data looks like, and what ''signal'' we are measuring. Download the data file named <tt>'fall2019_StudentData_3.mat'</tt> from the course dropbox folder. This is raw data that was collected by 20.309 students in the Fall of 2019. Load the file into your MATLAB workspace, and you should see a variable called <tt>yeastOsmoticShockData</tt>. This is a struct which contains the movie data, along with some other relevant experimental parameters:<br />
<pre><br />
>> yeastOsmoticShockData<br />
<br />
yeastOsmoticShockData = <br />
<br />
struct with fields:<br />
<br />
Movie: [544×728×2×32 uint16]<br />
Time: [32×2 double]<br />
ValveState: [32×1 logical]<br />
ValveOscillationPeriod: 480<br />
BlueCameraGainAndExposure: [3 5000000]<br />
GreenCameraGainAndExposure: [15 5000000]<br />
</pre><br />
<br />
Notice that the movie contains two colors (the third dimension of the movie has a length of 2). The movie matrix is constructed so that the first color represents the GFP-Hog1 signal, and the second color represents the nuclear signal (tagged with RFP). <br />
<br />
Use <tt>implay</tt> to watch each color of the movie:<br />
<pre><br />
implay(double(yeastOsmoticShockData.Movie(:,:,1,:))/4095);<br />
implay(double(yeastOsmoticShockData.Movie(:,:,2,:))/4095);<br />
</pre><br />
Do they behave as you would expect?<br />
<br />
{{Template:Assignment Turn In|message =<br />
# Step through the frames of the movie using the implay controls. Identify a frame where the signal is "high" - in other words, where the Hog1 signal is localized in the nucleus. Turn in the frame number that you've identified and a screen shot of the GFP-Hog1 movie at that frame number.<br />
# Repeat for a frame where the signal is "low" - in other words, where the Hog1 signal is uniformly distributed throughout the cell. Turn in the frame number that you've identified and a screen shot of the GFP-Hog1 movie at that frame number.<br />
}}<br />
<br />
{{Template:20.309 bottom}}</div>Juliesuttonhttp://measurebiology.org/wiki/Spring_2020_Assignment_8Spring 2020 Assignment 82020-04-27T19:09:52Z<p>Juliesutton: /* Fourier transform table */</p>
<hr />
<div>{{Template: 20.309}}<br />
==Circuit analogies==<br />
{{Template:Assignment Turn In|message= <br />
For each of the systems below, find an analogous circuit.<br />
}}<br />
<br />
<gallery><br />
File:Thermal System Analogy Problem.png |Thermal system:Coffee in a thermos<br />
File:Mechanical System Analogy Problem.png|Mechanical system: mass and damper<br />
</gallery><br />
<br />
==Convolution practice==<br />
{{Template:Assignment Turn In|message= <br />
For each of the pairs of functions below, plot the convolution of the two functions, <math>Y=A*B</math><br />
}}<br />
<br />
{| style="width: 85%;"<br />
!<math>A</math><br />
!<math>B</math><br />
!<math>Y=A*B</math><br />
|-<br />
|[[File:delta(t+1)+delta(t-1).png|250 px]]<br />
|[[File:delta(t+1)+delta(t-1).png|250 px]]<br />
|[[File:bare convolution axes.png|250 px]]<br />
|-<br />
|[[File:delta(t+1)+delta(t-1).png|250 px]]<br />
|[[File:box w=1.png|250 px]]<br />
|[[File:bare convolution axes.png|250 px]]<br />
|-<br />
|[[File:delta(t+1)+delta(t-1).png|250 px]]<br />
|[[File:box w=2.png|250 px]]<br />
|[[File:bare convolution axes.png|250 px]]<br />
|-<br />
|[[File:box w=1.png|250 px]]<br />
|[[File:box w=1.png|250 px]]<br />
|[[File:bare convolution axes.png|250 px]]<br />
|-<br />
|[[File:delta(t+1)+delta(t-1).png|250 px]]<br />
|[[File:triangle.png|250 px]]<br />
|[[File:bare convolution axes.png|250 px]]<br />
|-<br />
|[[File:delta(t).png|250 px]]<br />
|[[File:triangle.png|250 px]]<br />
|[[File:bare convolution axes.png|250 px]]<br />
|}<br />
<br />
<br />
==Fourier transform table==<br />
The two tables below show important properties of the Fourier transform and several useful transform pairs. You can use the tables of pairs and properties to figure out the transforms of an endless number of functions. <br />
<br />
{{Template:Assignment Turn In|message=<br />
<ol type="A"><br />
<li>For each of the named functions in the ''Table of Common Functions and their Fourier Transforms'', sketch the function in the time domain as well as the magnitude of its Fourier transform. Show relevant constants (for example: ''a'', <math>\alpha</math>, and <math>f_0</math>).</li><br />
<li>Sketch the transform of <math>\cos^4(\omega_0 t)</math>.</li><br />
<li>Sketch the magnitude of the fourier transform of <math>e^{-\alpha t} u(t) \times \cos(\omega_0 t)</math>. Assume <math>\alpha\ll\omega_0</math>.</li><br />
}}<br />
<br />
<center><br />
[[Image: FourierTransformsTable.png|500 px|<caption>Short table of Fourier transform properties</caption>]]<br />
[[Image: TimeFrequencyDomains_MoreTransformPairsTable.png|500 px|<caption>Short table of Fourier transform pairs</caption>]]<br />
</center><br />
<br />
{{Template: 20.309 bottom}}</div>Juliesuttonhttp://measurebiology.org/wiki/File:TimeFrequencyDomains_MoreTransformPairsTable.pngFile:TimeFrequencyDomains MoreTransformPairsTable.png2020-04-24T20:17:53Z<p>Juliesutton: Juliesutton uploaded a new version of &quot;File:TimeFrequencyDomains MoreTransformPairsTable.png&quot;</p>
<hr />
<div></div>Juliesuttonhttp://measurebiology.org/wiki/Spring_2020_Assignment_9Spring 2020 Assignment 92020-04-22T20:54:10Z<p>Juliesutton: Created page with "Category:20.309 Category:Electronics {{Template:20.309}} __NOTOC__ ==Written questions== {{Template:Assignment Turn In|message = Read the paper and answer the writte..."</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:Electronics]]<br />
{{Template:20.309}}<br />
__NOTOC__<br />
==Written questions==<br />
{{Template:Assignment Turn In|message = <br />
<br />
Read the paper and answer the written questions in [[Assignment 9 Overview: Analyzing yeast images]].<br />
<br />
}}<br />
<br />
==Yeast image analysis code==<br />
<br />
Download the data provided in the class dropbox folder. This data was collected by 20.309 students last semester. The movies were collected using the same 20.309 microscope that you built in class with an upgrade (that you would have done in Assignment 8 had we been on campus...) to incorporate a second fluorescence color. During an experiment, the microscope first recorded an image with blue illumination (exciting GFP) followed by one with green illumination (exciting RFP). It had a 40x objective and a 125 mm tube lens - remember that results in a 25x magnification. <br />
<br />
The sample is similar to the one in Mettetal ''et al.'': we are using ''S. cerevisiae'' with the protein Hog1 fused to GFP (which is excited by blue light) and an mRNA binding protein (we'll call MCP) fused to tagRFP (which is excited by green light) to locate the nucleus. A solenoid valve controls whether or not the medium provided to the yeast cells contains a high salt concentration, and in the experiment we oscillate between a high and low salt medium at a fixed rate. We expect to see the yeast cells 'respond' to an osmotic shock as a more localized Hog1-GFP signal that overlaps with the signal from the nucleus (RFP). Our goal is to calculate the average correlation coefficient between Hog1 and the nucleus as a function of the high/low valve state.<br />
<br />
Load some data into MATLAB:<br />
<pre><br />
load('fall2019_StudentData_1');<br />
</pre><br />
The data is stored in a struct called <tt>yeastOsmoticShockData</tt>. <br />
<pre> <br />
yeastOsmoticShockData = <br />
<br />
struct with fields:<br />
<br />
Movie: [544×728×2×16 uint16]<br />
Time: [16×2 double]<br />
ValveState: [16×2 logical]<br />
ValveOscillationPeriod: 960<br />
BlueCameraGainAndExposure: [6 5000000]<br />
GreenCameraGainAndExposure: [15 5000000]<br />
</pre><br />
<br />
The data structure contains all the important information from the experiment:<br />
* <tt>yeastOsmoticShockData.Movie</tt> is a two-color movie, where the 3d dimension is the color (1 = Blue excitation = GFP, 2 = Green excitation = RFP), and the fourth is the frame number. Notice that <tt> yeastOsmoticShockData.Movie</tt> is a uint16 data type which is the same as the movies you worked with in the particle tracking assignments. <br />
* <tt>yeastOsmoticShockData.Time</tt> is the time stamp of each frame, recorded in seconds from the start of the experiment. Column 1 contains the timestamps of the GFP images, column 2 is for the RFP images.<br />
* <tt>yeastOsmoticShockData.ValveOscillationPeriod</tt> is the oscillation valve period, in seconds.<br />
* <tt>yeastOsmoticShockData.BlueCameraGainAndExposure</tt> and <tt>yeastOsmoticShockData.GreenCameraGainAndExposure</tt> are the blue and green illumination camera settings in the format <tt>[gain, exposure]</tt>, where <tt>exposure</tt> is in microseconds.<br />
* <tt>yeastOsmoticShockData.ValveState</tt> is the state of the solenoid pinch valve controlling the salt concentration at the time of each image. The valve state is 0 for low salt, and 1 for high salt. If you need to know the valve state more precisely, it can be calculated with the following conditional statement: <math> sin(2 \pi t/T) \ge 0 </math>, where ''T'' is the valve oscillation period, and ''t'' is the time since the start of the experiment (this is how the state is determined in the software).<br />
<br />
{{Template:Assignment Turn In|message = Choose a single frame of the movie. Make a four panel figure and display: the GFP image, the RFP image (both with a scale bar), and their corresponding image histograms. Include an appropriate title for each panel. }}<br />
<br />
==Analyze a frame==<br />
<br />
In each frame of our movie, we need to locate and identify individual cells, and find the correlation coefficient between the cell image and the nuclei image. If the Hog1 signal is localized in the nucleus, the correlation should be close to 1, while if there is no correlation between the signals, the correlation will be close to zero. In reality, we'll likely see correlations close to about 0.5, but we'll look for an increase or decrease in the signal to determine the amount of Hog1 localization. <br />
<br />
[[Image:FindingCellsAndNucleiExample.png|center|thumb|400px]]<br />
<br />
Start with the following basic layout of the function:<br />
<br />
<pre><br />
function [AverageNucleusHog1Correlation, StandardErrorNucleusHog1Correlation] = AnalyzeFrame( TwoColorFrame, BackgroundImage, BackgroundRectangle )<br />
<br />
% 1. estimate the background level<br />
backgroundScaleFactors = FindBackgroundScaleFactor(TwoColorFrame,BackgroundImage, BackgroundRectangle);<br />
<br />
cellFrame = TwoColorFrame(:,:,1) - backgroundScaleFactors(1)*BackgroundImage(:,:,1);<br />
nucleiFrame = TwoColorFrame(:,:,2) - backgroundScaleFactors(2)*BackgroundImage(:,:,2);<br />
<br />
% 2. identify individual yeast cells in the GFP image<br />
% 3. calculate the correlation coefficient between the GFP intensities an RFP intensities within each cell region<br />
% 4. output the mean correlation coefficient for all cells in the frame<br />
<br />
end<br />
</pre><br />
<br />
As you work through this part of the Assignment, fill out the <tt>AnalyzeFrame</tt> function to incorporate each of the outlined steps. <br />
<br />
===Finding cells===<br />
<br />
We could consider using a threshold to identify cells in an image, similar to how we found fluorescent particles in Assignments 4 and 5. The problem here, is that thresholding doesn't allow us separate a single cells from a clump of cells. Since we know that yeast cells are roughly spherical, we can use <tt>imfindcircles</tt> to locate them. <tt>imfindcircles</tt> is an implemenattion of the [https://www.mathworks.com/help/images/ref/imfindcircles.html| Hough transform]. <br />
<br />
<pre><br />
[cellCenters, cellRadii] = imfindcircles(cellFrame,[Rmin Rmax],'ObjectPolarity','bright','Sensitivity',0.95);<br />
</pre><br />
<br />
Here, <tt>Rmin</tt> and <tt>Rmax</tt> are the smallest and largest expected 'radii'. How can you estimate <tt>Rmin</tt> and <tt>Rmax</tt>?<br />
<br />
The function <tt>viscircles(cellCenters,cellRadii)</tt> may be useful to visualize your results.<br />
<br />
=== Loop through each cell and calculate the correlation coefficient between GFP and RFP ===<br />
<br />
Next we want to calculate the response of the Hog1 protein. In the paper, they calculated the response as the ratio of GFP intensities inside and outside the nucleus: <math> R_{Hog1} = \frac{<GFP_{nucleus}>}{<GFP_{cell}>}</math>. We'll use an alternative way to find the response: by finding the correlation coefficient between the GFP and RFP signals within each cell.<br />
<br />
Finish filling out <tt>AnalyzeFrame</tt> to loop through each circle found in the frame and extract the corresponding pixel intensities from your GFP and RFP images. Then, calculate the correlation coefficients between these two intensity vectors (<tt>corr</tt> in MATLAB). The following helper function may be useful. <br />
<pre><br />
function Im = createMaskFromCircles(centers,radii,ImageSize)<br />
Im = zeros(ImageSize);<br />
[X, Y] = meshgrid(1:ImageSize(2), 1:1:ImageSize(1));<br />
<br />
for ii = 1:length(radii)<br />
isBright = (X-centers(ii,1)).^2+(Y-centers(ii,2)).^2 <= radii(ii,1)^2;<br />
isAlreadyFound = (Im == 1);<br />
Im = Im + (isBright & ~isAlreadyFound);<br />
end<br />
Im( Im > 1 ) = 1;<br />
end<br />
</pre><br />
<br />
Once you've collected each individual correlation coefficient, find the mean and standard error, and output these from the function.<br />
<br />
== Analyze the movie==<br />
<br />
Once you've set up your analysis for a single frame, you'll now want to loop through the movie and collect the responses as a function of time.<br />
<br />
{{Template:Assignment Turn In|message = <br />
<br />
# Analyze each frame of the movie and plot the average cellular response as a function of time. <br />
#* In a single figure, plot the data in one subplot, and the pinch valve state (1 for high salt, 0 for low salt) in a second subplot. <br />
# Write a function to fit the response to a sinusoid and extract the predicted amplitude and phase shift of the response signal. Plot the best fit function on the same plot as your data. <br />
# Report the resulting best fit parameters. }}<br />
<br />
<br />
{{Template:Assignment Turn In|message = Turn in all your MATLAB code in pdf format. No need to include functions that you used but did not modify.}}<br />
{{Template:Assignment 9 Analyzing yeast images navigation}}<br />
{{Template:20.309 bottom}}</div>Juliesuttonhttp://measurebiology.org/wiki/Spring_2020_Assignment_8Spring 2020 Assignment 82020-04-20T16:02:09Z<p>Juliesutton: /* Circuit analogies */</p>
<hr />
<div>{{Template: 20.309}}<br />
==Circuit analogies==<br />
{{Template:Assignment Turn In|message= <br />
For each of the systems below, find an analogous circuit.<br />
}}<br />
<br />
<gallery><br />
File:Thermal System Analogy Problem.png |Thermal system:Coffee in a thermos<br />
File:Mechanical System Analogy Problem.png|Mechanical system: mass and damper<br />
</gallery><br />
<br />
{{:Assignment 8, Part 0: convolution practice}}<br />
<br />
{{Template:20.309 bottom}}<br />
{{Template: 20.309 bottom}}</div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_8,_Part_0:_convolution_practiceAssignment 8, Part 0: convolution practice2020-04-20T16:01:01Z<p>Juliesutton: </p>
<hr />
<div><noinclude><br />
<br />
[[Category:20.309]]<br />
[[Category:Lab Manuals]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
</noinclude><br />
<br />
The following two tables will pop up frequently in 20.309 for the rest of the semester. Table 8.0.1 describes the Fourier transform and many of its useful properties, while table 8.0.2 contains the transform pairs of many common functions. These two tables are useful because you can combine functions in table 8.0.2 using the properties in table 8.0.1 to figure out the transforms of an endless number of functions (without doing any math!). <br />
<br />
[[Image: FourierTransformsTable.png|thumb|left|500 px|<caption>Table 8.0.1: Short table of Fourier transform properties</caption>]]<br />
<br />
[[Image: TimeFrequencyDomains_MoreTransformPairsTable.png|thumb|left|500 px|<caption>Table 8.0.2: Short table of Fourier transform pairs</caption>]]<br />
<br />
{{Template:Assignment Turn In|message=<br />
<br />
<ol type="A"><br />
<br />
<li>Sketch each function in table 8.0.2 as well as the magnitude of its Fourier transform. Include any relevant constants in your sketch (for example: ''a'', <math>\alpha</math>, and <math>f_0</math>).<br />
</li><br />
<br><br />
<li><br />
In class we found the Fourier transform of <math>\cos^2(\omega_0 t)</math>. Use graphical convolution to determine the transform of <math>\cos^4(\omega_0 t)</math>.<br />
</li><br />
<br><br />
<li><br />
Using the transform pairs in table 8.0.2, sketch the fourier transform of <math>e^{-\alpha t} u(t) \times \cos(\omega_0 t)</math>. Assume that <math>\alpha\ll\omega_0</math>.<br />
</li><br />
<br><br />
<li> Table 8.0.3 shows plots of eight time-domain signals A-H. The table on the right includes magnitude plots of the Fourier transform of ten signals numbered 1-10. For each time domain signal A-H, write the number 1-10 in the empty column of the matching frequency-domain signal. You may use a numbered plot more than once.<br><br />
Some of the frequency plots are shown on log-log axes and some are linear, as indicated by the plot title.<br><br />
''Hint:'' Gaussian or white noise is a random signal with equal contributions from ''every frequency''.<br />
<br />
</li><br />
<br><br />
<br />
</ol>}}<br />
<br />
[[Image: PSet4_ConvolutionImage.png|thumb|center|500 px|<caption>Table 8.0.3</caption>]]<br />
<br />
<noinclude><br />
{{Template:Assignment 8 flow channel & two-color microscope navigation}}<br />
<br />
{{Template:20.309 bottom}}<br />
</noinclude></div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_8,_Part_0:_convolution_practiceAssignment 8, Part 0: convolution practice2020-04-20T15:57:48Z<p>Juliesutton: </p>
<hr />
<div><noinclude><br />
<br />
[[Category:20.309]]<br />
[[Category:Lab Manuals]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
</noinclude><br />
<br />
The two tables below will pop up frequently in 20.309 for the rest of the semester. Table 8.0.1 describes the Fourier transform and many of its useful properties, while table 8.0.2 contains the transform pairs of many common functions. These two tables are useful because you can combine functions in table 8.0.2 using the properties in table 8.0.1 to figure out the transforms of an endless number of functions (without doing any math!). <br />
<br />
[[Image: FourierTransformsTable.png|thumb|center|500 px|<caption>Table 8.0.1: Short table of Fourier transform properties</caption>]]<br />
<br />
[[Image: TimeFrequencyDomains_MoreTransformPairsTable.png|thumb|center|500 px|<caption>Table 8.0.2: Short table of Fourier transform pairs</caption>]]<br />
<br />
{{Template:Assignment Turn In|message=<br />
<br />
<ol type="A"><br />
<br />
<li>Sketch each function in table 8.0.2 as well as the magnitude of its Fourier transform. Include any relevant constants in your sketch (for example: ''a'', <math>\alpha</math>, and <math>f_0</math>).<br />
</li><br />
<br><br />
<li><br />
In class we found the Fourier transform of <math>\cos^2(\omega_0 t)</math>. Use graphical convolution to determine the transform of <math>\cos^4(\omega_0 t)</math>.<br />
</li><br />
<br><br />
<li><br />
Using the transform pairs in table 8.0.2, sketch the fourier transform of <math>e^{-\alpha t} u(t) \times \cos(\omega_0 t)</math>. Assume that <math>\alpha\ll\omega_0</math>.<br />
</li><br />
<br><br />
<li> Table 8.0.3 shows plots of eight time-domain signals A-H. The table on the right includes magnitude plots of the Fourier transform of ten signals numbered 1-10. For each time domain signal A-H, write the number 1-10 in the empty column of the matching frequency-domain signal. You may use a numbered plot more than once.<br><br />
Some of the frequency plots are shown on log-log axes and some are linear, as indicated by the plot title.<br><br />
''Hint:'' Gaussian or white noise is a random signal with equal contributions from ''every frequency''.<br />
<br />
</li><br />
<br><br />
<br />
</ol>}}<br />
<br />
[[Image: PSet4_ConvolutionImage.png|thumb|center|500 px|<caption>Table 8.0.3</caption>]]<br />
<br />
<noinclude><br />
{{Template:Assignment 8 flow channel & two-color microscope navigation}}<br />
<br />
{{Template:20.309 bottom}}<br />
</noinclude></div>Juliesuttonhttp://measurebiology.org/wiki/File:TimeFrequencyDomains_MoreTransformPairsTable.pngFile:TimeFrequencyDomains MoreTransformPairsTable.png2020-04-20T15:51:06Z<p>Juliesutton: Juliesutton uploaded a new version of &quot;File:TimeFrequencyDomains MoreTransformPairsTable.png&quot;</p>
<hr />
<div></div>Juliesuttonhttp://measurebiology.org/wiki/File:FourierTransformsTable.pngFile:FourierTransformsTable.png2020-04-20T15:50:14Z<p>Juliesutton: Juliesutton uploaded a new version of &quot;File:FourierTransformsTable.png&quot;</p>
<hr />
<div></div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_8,_Part_0:_convolution_practiceAssignment 8, Part 0: convolution practice2020-04-20T15:49:51Z<p>Juliesutton: </p>
<hr />
<div><noinclude><br />
[[Category:20.309]]<br />
[[Category:Lab Manuals]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
</noinclude><br />
<br />
The two tables below will pop up frequently in 20.309 for the rest of the semester. Table 8.0.1 describes the Fourier transform and many of its useful properties, while table 8.0.2 contains the transform pairs of many common functions. These two tables are useful because you can combine functions in table 8.0.2 using the properties in table 8.0.1 to figure out the transforms of an endless number of functions (without doing any math!). <br />
<br />
[[Image: FourierTransformsTable.png|thumb|center|500 px|<caption>Table 8.0.1: Short table of Fourier transform properties</caption>]]<br />
<br />
[[Image: TimeFrequencyDomains_MoreTransformPairsTable.png|thumb|center|500 px|<caption>Table 8.0.2: Short table of Fourier transform pairs</caption>]]<br />
<br />
{{Template:Assignment Turn In|message=<br />
<br />
<ol type="A"><br />
<br />
<li>Sketch each function in table 8.0.2 as well as the magnitude of its Fourier transform. Include any relevant constants in your sketch (for example: ''a'', <math>\alpha</math>, and <math>f_0</math>).<br />
</li><br />
<br><br />
<li><br />
In class we found the Fourier transform of <math>\cos^2(\omega_0 t)</math>. Use graphical convolution to determine the transform of <math>\cos^4(\omega_0 t)</math>.<br />
</li><br />
<br><br />
<li><br />
Using the transform pairs in table 8.0.2, sketch the fourier transform of <math>e^{-\alpha t} u(t) \times \cos(\omega_0 t)</math>. Assume that <math>\alpha\ll\omega_0</math>.<br />
</li><br />
<br><br />
<li> Table 8.0.3 shows plots of eight time-domain signals A-H. The table on the right includes magnitude plots of the Fourier transform of ten signals numbered 1-10. For each time domain signal A-H, write the number 1-10 in the empty column of the matching frequency-domain signal. You may use a numbered plot more than once.<br><br />
Some of the frequency plots are shown on log-log axes and some are linear, as indicated by the plot title.<br><br />
''Hint:'' Gaussian or white noise is a random signal with equal contributions from ''every frequency''.<br />
<br />
</li><br />
<br><br />
<br />
</ol>}}<br />
[[Image: PSet4_ConvolutionImage.png|thumb|center|500 px|<caption>Table 8.0.3</caption>]]<br />
<br />
<noinclude><br />
{{Template:Assignment 8 flow channel & two-color microscope navigation}}<br />
<br />
{{Template:20.309 bottom}}<br />
</noinclude></div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_8,_Part_0:_convolution_practiceAssignment 8, Part 0: convolution practice2020-04-20T14:32:05Z<p>Juliesutton: </p>
<hr />
<div><noinclude><br />
[[Category:20.309]]<br />
[[Category:Lab Manuals]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
</noinclude><br />
<br />
{{Template:Assignment Turn In|message=Turn in your answers to the following questions}}<br />
<br />
You may find the Fourier transform Tables 8.0.1 and 8.0.2 useful. Note that there are a few functions that you may not have seen before including:<br />
* ''u(t)'' is the unit step function <math>u(t) =<br />
\begin{cases}<br />
0, & \text{if }t<0 \\<br />
1, & \text{if }t\geq0<br />
\end{cases}<br />
</math><br />
* sinc''(ax)'' is defined as: <math> \text{sinc}(ax) = \frac{\sin(ax)}{ax}</math><br />
* rect''(ax)'' is the box function: <math> \text{rect}(ax) =<br />
\begin{cases}<br />
0, & \text{if } |ax|> 1/2 \\<br />
1, & \text{if } |ax| \leq 1/2<br />
\end{cases}<br />
</math><br />
<br />
[[Image: FourierTransformsTable.png|thumb|center|500 px|<caption>Table 8.0.1: Short table of Fourier transform properties</caption>]]<br />
[[Image: TimeFrequencyDomains_MoreTransformPairsTable.png|thumb|center|500 px|<caption>Table 8.0.2: Short table of Fourier transform pairs</caption>]]<br />
<br />
<br />
<ol type="A"><br />
<li><br />
In class we found the Fourier transform of <math>\cos^2(\omega_0 t)</math>. Use graphical convolution to determine the transform of <math>\cos^4(\omega_0 t)</math>.<br />
</li><br />
<br><br />
<li><br />
Using the transform pairs in table 8.0.2, sketch the fourier transform of <math>e^{-\alpha t} u(t) \times \cos(\omega_0 t)</math>. Assume that <math>\alpha\ll\omega_0</math>.<br />
</li><br />
<br><br />
<li> Table 8.0.3 shows plots of eight time-domain signals A-H. The table on the right includes magnitude plots of the Fourier transform of ten signals numbered 1-10. For each time domain signal A-H, write the number 1-10 in the empty column of the matching frequency-domain signal. You may use a numbered plot more than once.<br><br />
Some of the frequency plots are shown on log-log axes and some are linear, as indicated by the plot title.<br><br />
<br />
[[Image: PSet4_ConvolutionImage.png|thumb|center|500 px|<caption>Table 8.0.3</caption>]]<br />
</li><br />
<br><br />
<br />
</ol><br />
<br />
<noinclude><br />
{{Template:Assignment 8 flow channel & two-color microscope navigation}}<br />
<br />
{{Template:20.309 bottom}}<br />
</noinclude></div>Juliesuttonhttp://measurebiology.org/wiki/20.309_Main_Page20.309 Main Page2020-04-20T14:20:25Z<p>Juliesutton: /* Module 2: electronics, signals and systems */</p>
<hr />
<div>{{Template:20.309}}<br />
__NOTOC__<br />
Bioinstrumentation scholars: you are welcome to edit and improve this course wiki. Correct, helpful, exquisitely-expressed changes will increase your class participation grade. Alterations not meeting those critera may have the opposite effect.<br />
<br />
If you would like to contribute to this site, please [[Special:RequestAccount|request an account]].<br />
<br />
== 20.309 course information==<br />
<br />
* [[20.309:Course Information| 20.309 course information]]<br />
* [[20.309:Safety| lab safety]]<br />
* Reserve a lab station here: [http://measurebiology.org/reservations/Web/schedule.php Bioinstrumentation Lab reservation system]<br />
<br />
== 20.309 master schedule==<br />
<br />
<div style="padding: 10px; width: 740px; border: 5px solid #000FFF;"><br />
===Module 1: Optics & Microscopy===<br />
{| border=1px<br />
|style="width: 10%;"|'''Important dates''' <br />
|style="width: 15%;"|'''Assignment due'''<br />
|style="width: 30%;"|'''Assignment page links'''<br />
|style="width: 45%;"|'''Useful resources'''<br />
|-- <br />
| <font color=#04B45F>2/16/20</font color><br />
| Assignment 1<br />
| <br />
[[Assignment 1 Overview: Transillumination microscopy| Assignment 1 Overview]]<br />
* [[Assignment 1, Part 1: Pre-lab questions|Part 1: Pre-lab questions]]<br />
* [[Assignment 1, Part 2: Optics bootcamp|Part 2: Optics bootcamp]]<br />
* [[Assignment 1, Part 3: Building your transillumination microscope|Part 3: Build a microscope]]<br />
* [[Assignment 1, Part 4: Building your transilluminated microscope|Part 4: Measure stuff]]<br />
| <br />
* [https://stellar.mit.edu/S/course/20/sp20/20.309/materials.html Lectures 1 through 9 of the 20.309 class]<br />
* [http://www.microscopyu.com/articles/formulas/formulasri.html Snell's law]<br />
* [[Understanding log plots]]<br />
|-<br />
| <font color=#04B45F>2/23/20</font color><br />
| Assignment 2<br />
| [[Assignment 2: fluorescence microscopy| Assignment 2 Overview]]<br />
* [[Assignment 2 Part 1: Noise in images|Part 1: Noise in images]]<br />
* [[Assignment 2 Part 2: Fluorescence microscopy|Part 2: Fluorescence microscopy]]<br />
* [[Assignment 2 Part 3: Build an epi-illuminator for your microscope|Part 3: Build an epi-illuminator for your microscope]]<br />
|<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [[Optical aberrations]]<br />
* [[Aperture and field stops]]<br />
* [[Optical detectors, noise, and the limit of detection]]<br />
* [[Manta G032 camera measurements]]<br />
|-<br />
| <font color=#04B45F>2/25/20</font color><br />
| Quiz 1 (in lecture)<br />
| ||<br />
|-<br />
| <font color=#04B45F>3/1/20</font color><br />
| Assignment 3<br />
| [[Assignment 3 Overview|Assignment 3 Overview]]<br />
* [[Assignment 3, Part 1: visualizing actin with fluorescence contrast|Part 1: fluorescence imaging]] <br />
* [[Assignment 3, Part 2: experimental design with fluorescence| Part 2: choosing filters]] <br />
|<br />
*[[Flat-field correction|Flat-field correction of non-uniform illumination]]<br />
*[[Matlab: Scalebars]]<br />
|-<br />
| <font color=#04B45F>3/8/20</font color><br />
| Assignment 4<br />
|{{Template:Assignment 4 navigation}}<br />
|<br />
* [http://www.microscopyu.com/articles/formulas/formulasresolution.html Resolution]<br />
* [[Physical optics and resolution]]<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[MATLAB: Estimating resolution from a PSF slide image]]<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
|-<br />
| <font color=#04B45F>4/3/20</font color><br />
| Assignment 5<br />
| [[Assignment 5: Spring 2020|Assignment 5 Overview (Spring 2020)]]<br />
* [[Assignment 5, Part 1: MSD difference tracking and microscope stability|Part 1: MSD difference tracking and microscope stability]] <br />
* [[Assignment 5, Part 2: live cell particle tracking of endocytosed beads| Part 2: Live cell particle tracking of endocytosed beads]]<br />
|<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
|-<br />
| <font color=#04B45F>4/7/20</font color><br />
| Exam 1 <br />
| <br />
|<br />
|-<br />
|}<br />
<br /><br />
<br />
===Module 2: electronics, signals and systems===<br />
{| border=1px<br />
|style="width: 10%;"|'''Important dates''' <br />
|style="width: 15%;"|'''Assignment due'''<br />
|style="width: 30%;"|'''Assignment page links'''<br />
|style="width: 45%;"|'''Useful resources'''<br />
|-- <br />
| <font color=#04B45F>4/13/20</font color><br />
| Assignment 6<br />
| [[Spring 2020 Assignment 6]]<br />
* (Previous semesters' assignment 6 for reference)<br />
* [[Assignment 6 Overview: two color microscope|Assignment 6 overview]]<br />
* [[Assignment 6, Part 1: build a two-color microscope]]<br />
* [[Assignment 6, Part 2: electronics written problems]]<br />
|<br />
* [[Electronics primer]]<br />
* [https://www.khanacademy.org/science/electrical-engineering/ee-circuit-analysis-topic Khan Academy Material, esp. "Ideal Circuit Elements" and "Voltage Divider"]<br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Input and output impedance]]<br />
|-<br />
| <font color=#04B45F>4/20/20</font color><br />
| Assignment 7<br />
| [[Spring 2020 Assignment 7]]<br />
* (Previous semesters' assignment 7 for reference)<br />
* [[Electronics boot camp I: passive circuits and transfer functions|Assignment 7 overview]]<br />
* [[Electronics written problems II|Assignment 7, Part 1: more electronics written problems]]<br />
* [[Electronics boot camp lab part 1|Assignment 7, Part 2: Electronics bootcamp I]]<br />
<br />
| <br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Bode plots|Transfer functions and Bode plots]]<br />
* [[Complex Number Review|Complex number review]]<br />
* [[Input and output impedance]]<br />
|-<br />
| <font color=#04B45F>4/21/20</font color><br />
| Quiz 2 (60 min)<br />
| <br />
|<br />
|-<br />
| <font color=#04B45F>4/27/20</font color><br />
| Assignment 8<br />
| [[Spring 2020 Assignment 8]]<br />
*(Previous semester's assignment 8 for reference)<br />
*[[Assignment 8 Overview: flow channel & two-color microscope|Assignment 8 Overview]]<br />
* [[Electronics bootcamp II: feedback systems|Part 1: feedback systems]]<br />
* [[Assignment 8, Part 2: fabricate a microfluidic device| Part 2: fabricate a microfluidic device]]<br />
* [[Assignment 8, Part 3: add flow control and test your device| Part 3: add flow control and test your device]]<br />
|[http://science.sciencemag.org/content/319/5862/482 *The* yeast paper: Mettelal ''et al.'', ''Science'' (2008)]<br />
|-<br />
| <font color=#04B45F>5/4/20</font color><br />
| Assignment 9<br />
| [[Assignment 9 Overview: Analyzing yeast images]]<br />
* [[Assignment 8, Part 0: convolution practice| Part 0: convolution practice]]<br />
* [[Assignment 9, Part 1: Analyze two-color yeast images| Part 1: Analyze two-color yeast images]]<br />
* [[Assignment 10, Part 1: Measuring the osmotic shock response of yeast| A10: Measure the osmotic shock response of yeast]]<br />
|<br />
|-<br />
| <font color=#04B45F>5/3/20</font color><br />
| Assignment 10 <br />
|(combined with Assignment 9 in Sp20)<br />
|<br />
|-<br />
| <font color=#04B45F>5/7/20</font color><br />
| Exam 2 (90 min)<br />
| <br />
|<br />
|-<br />
| cancelled<br />
| Assignment 11<br />
| [[Assignment 11: Tell us about your lab visit]]<br />
| <br />
|-<br />
|}<br />
</div><br />
<br />
==Optics and microscopy resources==<br />
===Background reading===<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [[Optical aberrations]]<br />
* [[Aperture and field stops]]<br />
* [[Optical detectors, noise, and the limit of detection]]<br />
* [[Manta G032 camera measurements]]<br />
* [[Understanding log plots]]<br />
<br />
===Code examples and simulations===<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[MATLAB: Estimating resolution from a PSF slide image]]<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
<br />
==Electronics resources==<br />
===Background reading===<br />
* [[Electronics primer]]<br />
* [https://www.khanacademy.org/science/electrical-engineering/ee-circuit-analysis-topic Khan Academy Material, esp. "Ideal Circuit Elements" and "Voltage Divider"]<br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Bode plots|Transfer functions and Bode plots]]<br />
* [[Complex Number Review|Complex number review]]<br />
* [[Input and output impedance]]<br />
<br />
==Limits of detection mini-lab==<br />
* [[Lab Manual: Limits of Detection]]<br />
* [[Lab Manual: Atomic Force Microscopy (AFM)]]<br />
* [[Limits of Detection: Data Sessions]]<br />
<br />
==MRI mini-lab==<br />
* [https://gate.nmr.mgh.harvard.edu/wiki/Tabletop_MRI/index.php/Main_Page MGH tabletop MRI]<br />
(Kept for historical reasons.)</div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_5,_Part_1:_MSD_difference_tracking_and_microscope_stabilityAssignment 5, Part 1: MSD difference tracking and microscope stability2020-04-01T19:38:53Z<p>Juliesutton: /* Develop sum and difference trajectory code */</p>
<hr />
<div>[[Category:Lab Manuals]]<br />
[[Category:20.309]]<br />
[[Category:Optical Microscopy Lab]]<br />
{{Template:20.309}}<br />
<br />
This is Part 1 of [[Assignment 5 Overview| Assignment 5]].<br />
<br />
[[File:How will the plots differ.jpg|center]]<br />
==Overview==<br />
In the last assignment, you made movies of particles as they[https://www.dancetime.com/dance-styles/jitterbug-dance/ jitterbugged] and [https://www.youtube.com/watch?v=oEEY1tq3-ak waltzed] through your microscope's field of view. You developed code to track each particle's unique steps, and you computed each particle's mean squared displacement (MSD), diffusion coefficient, and the implied viscosity of the solutions the particles were suspended in. In this assignment, you'll be tracking particles again. But this time the particles are inside of cells. The cells have been cultured with 0.84 &mu;m fluorescent microspheres, which enter the cells through the process of [https://en.wikipedia.org/wiki/Endocytosis endocytosis]. They are the same kind of cells you took pretty pictures of in assignment 3 (NIH 3T3). Trapped inside of the cells, the particles inside of cells will appear to be stationary to your naked eye. But they are definitely moving &mdash; albeit on a scale much smaller than the particles in glycerin solutions you measured previously.<br />
<br />
How do we know whether our microscope is even capable of measuring such small motions? Could we only be measuring artifacts (like vibrations of the microscope, or drifting of the stage)? <br />
<br />
By measuring the MSD of particles that have been glued down to a slide, we can actually diagnose what types of noise sources are contributing to our measurement, and thus what is the actual smallest motion (not due to noise) that we can measure. Some noise sources (like shot noise) are unavoidable, but others, (like mechanical vibrations and drift) we may be able to correct for.<br />
<br />
==MSD of sum and difference trajectories==<br />
{{:MSD of Sum and Difference Trajectories}}<br />
<br />
As you can see, measuring the MSD of the ''difference'' trajectories for two particles completely cancels out the mechanical noise. We can use the ''sum'' trajectories for two particles to diagnose the amount of mechanical noise initially present in your system: the difference between the MSDs of sum and difference trajectories is equal to (twice) the mechanical noise.<br />
<br />
==Develop sum and difference trajectory code==<br />
<br />
Modify your code from Assignment 4 to be able to track the ''sum'' and ''difference'' trajectories of particles in a movie.<br />
<br />
Steve actually generously shared his code with you!<br />
<pre><br />
function [ DifferenceTrajectories, SumTrajectories ] = CalculateSumAndDifferenceTrajectories( Trajectories )<br />
<br />
numberOfParticles = max( Trajectories(:,4) );<br />
allCombinations = nchoosek( 1:numberOfParticles, 2 );<br />
numberOfCombinations = size( allCombinations, 1 );<br />
<br />
SumTrajectories = cell( numberOfCombinations, 1 );<br />
DifferenceTrajectories = cell( numberOfCombinations, 1 );<br />
<br />
virtualParticleNumber = 1;<br />
<br />
for ii = 1:numberOfCombinations<br />
firstParticleIndex = allCombinations( ii, 1 );<br />
secondParticleIndex = allCombinations( ii, 2 );<br />
firstTrajectory = Trajectories( Trajectories(:, 4) == firstParticleIndex, : );<br />
secondTrajectory = Trajectories( Trajectories(:, 4) == secondParticleIndex, : );<br />
<br />
framesInCommon = intersect( firstTrajectory(:, 3), secondTrajectory(:, 3 ) );<br />
<br />
if( ~isempty( framesInCommon ) )<br />
firstTrajectory = firstTrajectory(ismember(firstTrajectory(:, 3 ), framesInCommon), :);<br />
secondTrajectory = secondTrajectory(ismember(secondTrajectory(:, 3 ), framesInCommon), :);<br />
thisDifference = (firstTrajectory - secondTrajectory)/sqrt(2);<br />
thisDifference(:,3) = framesInCommon';<br />
thisDifference(:,4) = virtualParticleNumber;<br />
DifferenceTrajectories{ii} = thisDifference;<br />
thisSum = (firstTrajectory + secondTrajectory)/sqrt(2);<br />
thisSum(:,3) = framesInCommon';<br />
thisSum(:,4) = virtualParticleNumber;<br />
SumTrajectories{ii} = thisSum;<br />
virtualParticleNumber = virtualParticleNumber + 1;<br />
end<br />
end<br />
<br />
DifferenceTrajectories = vertcat( DifferenceTrajectories{:} );<br />
SumTrajectories = vertcat( SumTrajectories{:} );<br />
<br />
end<br />
</pre><br />
<br />
===Sp2020 Update:===<br />
<br />
The above code finds the sum and difference trajectories for every possible pairing of particles (given by this line of code:<tt>allCombinations = nchoosek( 1:numberOfParticles, 2 );</tt>). This is a bit overkill, especially when the number of particles is more than 5 or 6. Instead, you can use the code below that Steve wrote, which takes the sum and difference of particle pairs that are closest to each other. It's not perfect, since it discards a particle if there is an odd number of them, but it's an improvement on <tt>CalculateSumAndDifferenceTrajectories</tt>, above. This function also only keeps trajectories that exist for the entire length of the movie which is helpful for filtering out 'bad' particles. <br />
<br />
If you have already completed assignment 5 using the old <tt>CalculateSumAndDifferenceTrajectories</tt> it's not a problem to keep using it, but know that you'll have many more traces on your MSD plot than necessary. <br />
<br />
<pre><br />
function [ DifferenceTrajectories, SumTrajectories ] = SumAndDifferenceTrajectoriesNearestNeighbor( Trajectories )<br />
<br />
numberOfFrames = max(Trajectories(:,3));<br />
[ counts, particleIds ] = groupcounts( Trajectories(:,4) );<br />
particleIds( counts ~= numberOfFrames ) = [];<br />
numberOfParticles = length( particleIds );<br />
<br />
if( numberOfParticles < 2 )<br />
DifferenceTrajectories = [];<br />
SumTrajectories = [];<br />
return<br />
end<br />
<br />
allPermutations = nchoosek( particleIds, numberOfParticles - mod( numberOfParticles, 2 ) );<br />
numberOfPermutations = size( allPermutations, 1 );<br />
initialPositions = Trajectories( Trajectories(:,3) == 1, : );<br />
totalDistanceSquared = zeros( 1, numberOfPermutations );<br />
<br />
for ii = 1:numberOfPermutations<br />
for jj = 1:2:size( allPermutations, 2 )<br />
firstParticlePosition = initialPositions(initialPositions(:,4) == allPermutations(ii,jj),1:2);<br />
secondParticlePosition = initialPositions(initialPositions(:,4) == allPermutations(ii,jj+1));<br />
totalDistanceSquared(ii) = totalDistanceSquared(ii) + sum( ( secondParticlePosition - firstParticlePosition ).^2 );<br />
end<br />
end<br />
[~, minimumDistancePermutation ] = min( totalDistanceSquared );<br />
bestPairing = allPermutations(minimumDistancePermutation,:);<br />
<br />
allPairings = reshape( bestPairing, [], 2 );<br />
<br />
numberOfCombinations = size( allPairings, 1 );<br />
SumTrajectories = cell( numberOfCombinations, 1 );<br />
DifferenceTrajectories = cell( numberOfCombinations, 1 );<br />
virtualParticleNumber = 1;<br />
<br />
for ii = 1:numberOfCombinations<br />
firstParticleIndex = allPairings( ii, 1 );<br />
secondParticleIndex = allPairings( ii, 2 );<br />
firstTrajectory = Trajectories( Trajectories(:, 4) == firstParticleIndex, : );<br />
secondTrajectory = Trajectories( Trajectories(:, 4) == secondParticleIndex, : );<br />
framesInCommon = intersect( firstTrajectory(:, 3), secondTrajectory(:, 3 ) );<br />
if( ~isempty( framesInCommon ) )<br />
firstTrajectory = firstTrajectory(ismember(firstTrajectory(:, 3 ), framesInCommon), :);<br />
secondTrajectory = secondTrajectory(ismember(secondTrajectory(:, 3 ), framesInCommon), :);<br />
thisDifference = firstTrajectory - secondTrajectory;<br />
thisDifference(:,3) = framesInCommon';<br />
thisDifference(:,4) = virtualParticleNumber;<br />
DifferenceTrajectories{ii} = thisDifference;<br />
thisSum = firstTrajectory + secondTrajectory;<br />
thisSum(:,3) = framesInCommon';<br />
thisSum(:,4) = virtualParticleNumber;<br />
SumTrajectories{ii} = thisSum;<br />
virtualParticleNumber = virtualParticleNumber + 1;<br />
end<br />
end<br />
<br />
DifferenceTrajectories = vertcat( DifferenceTrajectories{:} );<br />
SumTrajectories = vertcat( SumTrajectories{:} );<br />
<br />
</pre><br />
<br />
==Stability of microscope for particle tracking==<br />
[[Image:StabilityPlot2.jpg|right|400px|thumb|Your stability plot should look something like this.]]<br />
Once you have developed and tested your code, use the following procedure to measure the location precision of your microscope: <br />
<br />
# Bring a slide with fixed beads into focus. <br />
#*Choose a field of view in which you can see at least 3 beads using the 40× objective. <br />
#* Limit the field of view to only those beads by choosing a region of interest (ROI) in <tt>UsefulImageAcquisition</tt> tool. (Otherwise, you will have a gigantic movie that is hard to work with.)<br />
# Acquire a movie of beads for 3 minutes at a frame rate of at least 10 fps.<br />
# Use the code you developed to track the particles, and calculate the sum and difference trajectories. <br />
<br />
{{Template:Assignment Turn In|message =Plot the MSD versus time interval of <br />
# the sum trajectories, and<br />
# the difference trajectories.<br />
}}<br />
<br />
The motion of particles endocytosed by 3T3 cells requires an extremely stable microscope, otherwise the real motion of the beads will be obscured by noise. Your microscope will be considered stable enough if the MSD for the ''difference'' trajectory of two fixed particles starts out less than 500 nm<sup>2</sup> and remains less than 1000 nm<sup>2</sup> up to t = 180 s. If the MSD is larger than that, refine your apparatus and methodology until you achieve that goal.<br />
<br />
<br />
<br />
<br />
{{Template:Assignment 5 navigation}}<br />
{{Template:20.309 bottom}}</div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_5,_Part_2:_live_cell_particle_tracking_of_endocytosed_beadsAssignment 5, Part 2: live cell particle tracking of endocytosed beads2020-03-30T18:01:31Z<p>Juliesutton: </p>
<hr />
<div>[[Category:Lab Manuals]]<br />
[[Category:20.309]]<br />
[[Category:Optical Microscopy Lab]]<br />
{{Template:20.309}}<br />
<br />
This is Part 2 of [[Assignment 5 Overview| Assignment 5]].<br />
<br><br />
<br />
==Live cell particle tracking of endocytosed beads==<br />
<br />
We can also use particle tracking to probe cell samples. 0.84 μm diameter red fluorescent microspheres were mixed with the growth medium and added to the plated cells for a period of 12 to 24 hours for bead endocytosis. <br />
<br />
You will be given two plates of cells for these experiments. The cells must be imaged while they are alive, so the cells must be used the day you are given them:<br />
* <u>Dish 1</u> will be used to monitor particles in untreated cells, while <br />
* <u>Dish 2</u> will be reserved to track microspheres after adding CytoD.<br />
<br />
# Pre-warm your DMEM++ and CytoD to 37&deg;C<br />
# Carefully pipet out the medium from Dish 1. Gently rinse with 1mL of medium 2X to remove beads that were not endocytosed. Then, place 2 mL of fresh medium in dish. <br />
# Choose cells in Dish 1 with at least 2 but preferably 3 or 4 particles embedded in them and capture movies of the samples. Make sure to do this quickly, as the cells become unhealthy without the temperature and carbon dioxide regulation.<br />
#* By adjusting the LED current and exposure time, you should be able to use both bright field and fluorescence illumination simultaneously to find cells containing enough beads. Once you find a good-looking cell, turn off the LED and readjust the exposure time appropriately. <br />
#* Take as many movies as you can with about 2-5 particles in the field of view in each movie. <br />
# Next, carefully pipet out the medium in Dish 2. Gently rinse with 1mL of medium 2X to remove beads that were not endocytosed. <br />
# Treat the cells in Dish 2 with the cytoskeleton-modifying CytoD: Pipet out remaining medium, add 1 mL pre-warmed CytoD solution at 10 μM (pre-mixed for you) to the dish, and incubate for 15 minutes at 37&deg;C. It's a good idea to check on your cells after 15 minutes: sometimes they are in bad shape at that point but sometimes they still look very healthy. Wash 2X with 2 mL of pre-warmed DMEM++, leaving 2 mL in the dish when imaging.<br />
# Perform and repeat the particle tracking measurements again in Dish 2 as quickly as you are able. It would be good to image the beads in only one cell at a time, since different cells may have different degrees of cytoskeletal disruption. Take as many videos as you can before the cells become sad. The cells' physiology has now been significantly disrupted by the toxin CytoD, and they will die within a couple of hours.<br />
<br />
{{Template:Assignment Turn In|message=Turn in for particle tracking in cells:<br />
#Procedure<br />
#*Document the samples you prepared and used and how you captured images (camera settings including frame acquisition rate, number of frames, number of particles in the region of interest, choice of sample plane, etc)<br />
# Data<br />
#* Include a snapshot of the 0.84 &mu;m fluorescent beads monitored.<br />
#* Choose one untreated sample movie, and one Cyto-treated sample movie and plot two or more example bead trajectories from each movie. (Hint: If you subtract the initial position from each trajectory, then you can plot multiple trajectories on a single set of axes.)<br />
# Analysis and Results<br />
#* Plot the MSD vs time (from the difference trajectories) for untreated and Cyto D treated cells on a single set of log-log axes. Plot the individual MSDs from each pair of beads in each movie. Use a single color for all the untreated cells, and a different color for all the treated cells. Do not include MSDs of particles not endocytosed by the cells.<br />
#* Combine your data with others from the class to increase your sample size.<br />
# Discussion<br />
#* What kind of motion do you see described by your MSD vs &tau; results?<br />
#* What differences do you see between the untreated and Cyto D treated MSD curves? <br />
#* Please suggest an interpretation of the behavior of your cells based on your data.<br />
#* Include a thorough discussion of error sources and your approaches to minimize them. As in Assignment 4, list out the error sources in a table, including a category for the error source, type of error (random, systematic, fundamental, technical, etc.), the magnitude of the error, and a description and way to minimize each one. Your MSD measurements of the still beads (from Part 1 of this assignment) will be useful for estimating the magnitude of several significant error sources.<br />
<br />
Include any MATLAB code you've written as an appendix to your assignment.<br />
}}<br />
<br />
{{Template:Assignment 5 navigation}}<br />
{{Template:20.309 bottom}}</div>Juliesuttonhttp://measurebiology.org/wiki/Template:Assignment_8_flow_channel_%26_two-color_microscope_navigationTemplate:Assignment 8 flow channel & two-color microscope navigation2020-03-26T15:56:09Z<p>Juliesutton: /* Navigation */</p>
<hr />
<div>==Navigation==<br />
* [[Assignment 8 Overview: flow channel & two-color microscope|Overview]]<br />
* [[Electronics bootcamp II: feedback systems|Part 1: feedback systems]]<br />
* [[Assignment 8, Part 2: fabricate a microfluidic device| Part 2: fabricate a microfluidic device]] <br />
* [[Assignment 8, Part 3: add flow control and test your device| Part 3: add flow control and test your device]]<br />
<br />
Back to [[20.309 Main Page]]</div>Juliesuttonhttp://measurebiology.org/wiki/20.309_Main_Page20.309 Main Page2020-03-26T15:53:55Z<p>Juliesutton: /* Module 2: electronics, signals and systems */</p>
<hr />
<div>{{Template:20.309}}<br />
__NOTOC__<br />
Bioinstrumentation scholars: you are welcome to edit and improve this course wiki. Correct, helpful, exquisitely-expressed changes will increase your class participation grade. Alterations not meeting those critera may have the opposite effect.<br />
<br />
If you would like to contribute to this site, please [[Special:RequestAccount|request an account]].<br />
<br />
== 20.309 course information==<br />
<br />
* [[20.309:Course Information| 20.309 course information]]<br />
* [[20.309:Safety| lab safety]]<br />
* Reserve a lab station here: [http://measurebiology.org/reservations/Web/schedule.php Bioinstrumentation Lab reservation system]<br />
<br />
== 20.309 master schedule==<br />
<br />
<div style="padding: 10px; width: 740px; border: 5px solid #000FFF;"><br />
===Module 1: Optics & Microscopy===<br />
{| border=1px<br />
|style="width: 10%;"|'''Important dates''' <br />
|style="width: 15%;"|'''Assignment due'''<br />
|style="width: 30%;"|'''Assignment page links'''<br />
|style="width: 45%;"|'''Useful resources'''<br />
|-- <br />
| <font color=#04B45F>2/16/20</font color><br />
| Assignment 1<br />
| <br />
[[Assignment 1 Overview: Transillumination microscopy| Assignment 1 Overview]]<br />
* [[Assignment 1, Part 1: Pre-lab questions|Part 1: Pre-lab questions]]<br />
* [[Assignment 1, Part 2: Optics bootcamp|Part 2: Optics bootcamp]]<br />
* [[Assignment 1, Part 3: Building your transillumination microscope|Part 3: Build a microscope]]<br />
* [[Assignment 1, Part 4: Building your transilluminated microscope|Part 4: Measure stuff]]<br />
| <br />
* [https://stellar.mit.edu/S/course/20/sp20/20.309/materials.html Lectures 1 through 9 of the 20.309 class]<br />
* [http://www.microscopyu.com/articles/formulas/formulasri.html Snell's law]<br />
* [[Understanding log plots]]<br />
|-<br />
| <font color=#04B45F>2/23/20</font color><br />
| Assignment 2<br />
| [[Assignment 2: fluorescence microscopy| Assignment 2 Overview]]<br />
* [[Assignment 2 Part 1: Noise in images|Part 1: Noise in images]]<br />
* [[Assignment 2 Part 2: Fluorescence microscopy|Part 2: Fluorescence microscopy]]<br />
* [[Assignment 2 Part 3: Build an epi-illuminator for your microscope|Part 3: Build an epi-illuminator for your microscope]]<br />
|<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [[Optical aberrations]]<br />
* [[Aperture and field stops]]<br />
* [[Optical detectors, noise, and the limit of detection]]<br />
* [[Manta G032 camera measurements]]<br />
|-<br />
| <font color=#04B45F>2/25/20</font color><br />
| Quiz 1 (in lecture)<br />
| ||<br />
|-<br />
| <font color=#04B45F>3/1/20</font color><br />
| Assignment 3<br />
| [[Assignment 3 Overview|Assignment 3 Overview]]<br />
* [[Assignment 3, Part 1: visualizing actin with fluorescence contrast|Part 1: fluorescence imaging]] <br />
* [[Assignment 3, Part 2: experimental design with fluorescence| Part 2: choosing filters]] <br />
|<br />
*[[Flat-field correction|Flat-field correction of non-uniform illumination]]<br />
*[[Matlab: Scalebars]]<br />
|-<br />
| <font color=#04B45F>3/8/20</font color><br />
| Assignment 4<br />
|{{Template:Assignment 4 navigation}}<br />
|<br />
* [http://www.microscopyu.com/articles/formulas/formulasresolution.html Resolution]<br />
* [[Physical optics and resolution]]<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[MATLAB: Estimating resolution from a PSF slide image]]<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
|-<br />
| <font color=#04B45F>4/3/20</font color><br />
| Assignment 5<br />
| [[Assignment 5: Spring 2020|Assignment 5 Overview (Spring 2020)]]<br />
* [[Assignment 5, Part 1: MSD difference tracking and microscope stability|Part 1: MSD difference tracking and microscope stability]] <br />
* [[Assignment 5, Part 2: live cell particle tracking of endocytosed beads| Part 2: Live cell particle tracking of endocytosed beads]]<br />
|<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
|-<br />
| <font color=#04B45F>4/7/20</font color><br />
| Exam 1 <br />
| <br />
|<br />
|-<br />
|}<br />
<br /><br />
<br />
===Module 2: electronics, signals and systems===<br />
{| border=1px<br />
|style="width: 10%;"|'''Important dates''' <br />
|style="width: 15%;"|'''Assignment due'''<br />
|style="width: 30%;"|'''Assignment page links'''<br />
|style="width: 45%;"|'''Useful resources'''<br />
|-- <br />
| <font color=#04B45F>4/13/20</font color><br />
| Assignment 6<br />
| [[Assignment 6 Overview: two color microscope|Assignment 6 overview]]<br />
* [[Assignment 6, Part 1: build a two-color microscope]]<br />
* [[Assignment 6, Part 2: electronics written problems]]<br />
|<br />
* [[Electronics primer]]<br />
* [https://www.khanacademy.org/science/electrical-engineering/ee-circuit-analysis-topic Khan Academy Material, esp. "Ideal Circuit Elements" and "Voltage Divider"]<br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Input and output impedance]]<br />
|-<br />
| <font color=#04B45F>4/20/20</font color><br />
| Assignment 7<br />
| [[Electronics boot camp I: passive circuits and transfer functions|Assignment 7 overview]]<br />
* [[Electronics written problems II|Assignment 7, Part 1: more electronics written problems]]<br />
* [[Electronics boot camp lab part 1|Assignment 7, Part 2: Electronics bootcamp I]]<br />
<br />
| <br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Bode plots|Transfer functions and Bode plots]]<br />
* [[Complex Number Review|Complex number review]]<br />
* [[Input and output impedance]]<br />
|-<br />
| <font color=#04B45F>4/21/20</font color><br />
| Quiz 2 (60 min)<br />
| <br />
|<br />
|-<br />
| <font color=#04B45F>4/27/20</font color><br />
| Assignment 8<br />
| [[Assignment 8 Overview: flow channel & two-color microscope|Assignment 8 Overview]]<br />
* [[Electronics bootcamp II: feedback systems|Part 1: feedback systems]]<br />
* [[Assignment 8, Part 2: fabricate a microfluidic device| Part 2: fabricate a microfluidic device]]<br />
* [[Assignment 8, Part 3: add flow control and test your device| Part 3: add flow control and test your device]]<br />
|[http://science.sciencemag.org/content/319/5862/482 *The* yeast paper: Mettelal ''et al.'', ''Science'' (2008)]<br />
|-<br />
| <font color=#04B45F>5/4/20</font color><br />
| Assignment 9<br />
| [[Assignment 9 Overview: Analyzing yeast images]]<br />
* [[Assignment 8, Part 0: convolution practice| Part 0: convolution practice]]<br />
* [[Assignment 9, Part 1: Analyze two-color yeast images| Part 1: Analyze two-color yeast images]]<br />
* [[Assignment 10, Part 1: Measuring the osmotic shock response of yeast| A10: Measure the osmotic shock response of yeast]]<br />
|<br />
|-<br />
| <font color=#04B45F>5/3/20</font color><br />
| Assignment 10 <br />
|(combined with Assignment 9 in Sp20)<br />
|<br />
|-<br />
| <font color=#04B45F>5/7/20</font color><br />
| Exam 2 (90 min)<br />
| <br />
|<br />
|-<br />
| cancelled<br />
| Assignment 11<br />
| [[Assignment 11: Tell us about your lab visit]]<br />
| <br />
|-<br />
|}<br />
</div><br />
<br />
==Optics and microscopy resources==<br />
===Background reading===<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [[Optical aberrations]]<br />
* [[Aperture and field stops]]<br />
* [[Optical detectors, noise, and the limit of detection]]<br />
* [[Manta G032 camera measurements]]<br />
* [[Understanding log plots]]<br />
<br />
===Code examples and simulations===<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[MATLAB: Estimating resolution from a PSF slide image]]<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
<br />
==Electronics resources==<br />
===Background reading===<br />
* [[Electronics primer]]<br />
* [https://www.khanacademy.org/science/electrical-engineering/ee-circuit-analysis-topic Khan Academy Material, esp. "Ideal Circuit Elements" and "Voltage Divider"]<br />
* [[Impedance Analysis|Impedance analysis]]<br />
* [[Bode plots|Transfer functions and Bode plots]]<br />
* [[Complex Number Review|Complex number review]]<br />
* [[Input and output impedance]]<br />
<br />
==Limits of detection mini-lab==<br />
* [[Lab Manual: Limits of Detection]]<br />
* [[Lab Manual: Atomic Force Microscopy (AFM)]]<br />
* [[Limits of Detection: Data Sessions]]<br />
<br />
==MRI mini-lab==<br />
* [https://gate.nmr.mgh.harvard.edu/wiki/Tabletop_MRI/index.php/Main_Page MGH tabletop MRI]<br />
(Kept for historical reasons.)</div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_8_Overview:_flow_channel_%26_two-color_microscopeAssignment 8 Overview: flow channel & two-color microscope2020-03-26T15:51:32Z<p>Juliesutton: /* Overview */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:Electronics]]<br />
{{Template:20.309}}<br />
<br />
__NOTOC__<br />
<br />
==Overview==<br />
<br />
In Assignment 7, you familiarized yourselves with the basic tools and measurement techniques used in electronics. In the remaining assignments this semester, we'll work towards measuring the frequency-dependent osmotic shock response of yeast cells. We'll build a few circuits, but more importantly, we'll apply the framework that we've learned - using transfer functions, feedback, and Fourier transforms - to understanding a biological system. <br />
<br />
You'll start off this week's assignment 8 by examining the effects of gain on an electronic feedback system (Part 1). Then you'll build a microfluidic device and interface your microscope with computer controls for the LEDs and fluidic device (Part 2). Finally in Part 3, you'll validate that everything is working together by flowing fluorescent dyes through the device and recording how the fluorescence changes over time. <br />
<br />
# [[Electronics bootcamp II: feedback systems|Part 1: electronics bootcamp II]];<br />
# [[Assignment 8, Part 2: fabricate a microfluidic device| Part 2: fabricate a microfluidic device]]; <br />
# [[Assignment 8, Part 3: add flow control and test your device| Part 3: test your device with fluorescent dyes]].<br />
<br />
<br />
Submit your work on Stellar in a single PDF file with the naming convention <Lastname><Firstname>Assignment8.pdf.<br />
<br />
==References==<br />
<references /><br />
<br />
{{Template:Assignment 8 flow channel & two-color microscope navigation}}</div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_8,_Part_1:_fabricate_a_microfluidic_deviceAssignment 8, Part 1: fabricate a microfluidic device2020-03-26T15:50:59Z<p>Juliesutton: Juliesutton moved page Assignment 8, Part 1: fabricate a microfluidic device to Assignment 8, Part 2: fabricate a microfluidic device</p>
<hr />
<div>#REDIRECT [[Assignment 8, Part 2: fabricate a microfluidic device]]</div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_8,_Part_2:_fabricate_a_microfluidic_deviceAssignment 8, Part 2: fabricate a microfluidic device2020-03-26T15:50:58Z<p>Juliesutton: Juliesutton moved page Assignment 8, Part 1: fabricate a microfluidic device to Assignment 8, Part 2: fabricate a microfluidic device</p>
<hr />
<div>__NOTOC__<br />
<br />
==Overview==<br />
<br />
The remaining lab assignments will be based on work published in ''Science'' by MIT researchers in 2008 <ref>[http://science.sciencemag.org/content/319/5862/482 | J. T. Mettetal, D. Muzzey, C. Gomez-Uribe, and A. van Oudenaarden, "The Frequency Dependence of Osmo-Adaptation in Saccharomyces cerevisiae," Science, vol. 319, no. 5862, pp. 482–484, 2008]</ref>. The basic idea is that we will use a fluidic device to stimulate the yeast cells with high- or low-salt media, and measure the nuclear localization of the protein Hog1 using our fluorescence microscopes. We'll oscillate the flow of media from low to high salt at varying frequencies, and measure the amplitude and phase of Hog1's response. <br />
<br />
In this part of the assignment, you will make a microfluidic device that will allow you to switch the flow through a channel between two different fluid reservoirs. A previous version of the device used a method developed in Paul Blainey's lab to image the motion of transport molecules along DNA <ref>[https://www.jove.com/video/55923/a-simple-robust-high-throughput-single-molecule-flow-stretching-assay | K. Xiong and P. C. Blainey, “A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA,” J. Vis. Exp., no. 128, pp. 1–7, 2017]</ref>. In this iteration, a channel of any shape is cut out of double-sided tape and then sandwiched between a slab of PDMS and a cover slip. In the new version of the microfluidic device, the PDMS is poured over a 3-D printed master mold and then cured. The channels are sealed with a glass coverslip.<br />
<br />
PDMS (or polydimethylsiloxane) is a silicone elastomer made by mixing together a viscous liquid base with a crossliking agent. Once mixed and annealed at 60°C, the material will harden into a solid, rubber-like material that is optically clear, non-toxic, and chemically inert. Researchers typically use PDMS for microfluidics because they can use it to cast very small sharp features (down to ~1 micron), and they can covalently bond the PDMS to a glass coverslip, creating a sealed device that is readily compatible with most types of microscopy.<br />
<br />
Typically, researchers create a master mold by etching tiny features into silicon wafers. This process involves working in a clean room and handling some nasty chemicals. Once made, the silicon waver can be used repeatedly to cast new devices out of PDMS. (The wafers are very fragile, and they typically last until they are accidentally broken!) Thankfully, since our experiment does not require extremely small features, we can get away with using a 3-D printer to fabricate our master mold. We designed a Y-shaped channel with an 800 x 800 &mu;m cross section using CAD software, and sent the design out to a company called Protolabs for printing.<br />
<br />
==Cast a slab of PDMS from a 3-D printed master mold==<br />
<br />
Two notes before you begin:<br />
* This protocol has two steps with 15-60 minute wait times. Part 2 of this assignment can be completed in parallel with Part 1, if you want to keep making progress during the downtime.<br />
* PDMS is not harmful, but it is viscous and sticky. Work over a sheet of aluminum foil rather than directly on the work bench, wear gloves when pouring the elastomer base and crosslinking agent, and change them before touching other lab equipment. If you spill any PDMS, clean it up right away with a paper towel or kimwipe. <br />
* Work on a cutting mat, and not the bench, when punching holes and cutting tubing.<br />
<br />
Onward!<br />
<br />
[[Image: PDMSmaster.png|thumb|right|400 px|<caption>PDMS master mold.</caption>]]<br />
<br />
===Cast PDMS===<br />
# If not already on, turn the oven on to heat to 60°C.<br />
# Pour 9 g PDMS base into a plastic cup.<br />
# Carefully pour 1 g of the crosslinker into the same cup, being careful to note that it is far less viscous than the base.<br />
# Use a plastic stirrer to mix the crosslinker and base together really well. Stir for at least 2 minutes.<br />
# Pour the mixed PDMS into a 3D printed master mold. The mold will make 3 devices.<br />
# Degas the poured PDMS in the vacuum desiccator for at least 15 minutes, or until all bubbles are removed.<br />
# Bake at 60°C for 1 hour. <br />
<br />
===Unmold the cured PDMS and punch inlet and outlet holes ===<br />
# Using a ceramic blade, carefully cut around the inner edge of the 3D printed mold to separate it from the PDMS. Gently pry the PDMS away from the walls with the blade and carefully peel it off of the 3D printed mold.<br />
# Place the cured PDMS with the molded channels facing up on a clean cutting mat. <br />
# Use the 1 mm biopsy punch to make the two inlet and one outlet holes at each end of the Y-shape. Each time you push the punch through the PDMS, make sure to remove the core. <br />
# Inspect the device to ensure that each hole is cleanly formed.<br />
# Store the PDMS in a clean petri dish with lid on to prevent too much dust from sticking to the surface.<br />
# Repeat the punching steps for your remaining two devices.<br />
<br />
===Seal flow channels with a glass cover slip===<br />
# On a clean, clutter free section of the bench, lay out a 22 x 40 mm glass coverslip and the molded PDMS device (channel side up).<br />
# With supervision from an instructor, turn on the corona generator and pass it back and forth over the PDMS and coverslip for about 30 s.<br />
# Turn off the corona generator, then invert the PDMS onto the coverslip, trying to align the edges of the glass and PDMS as best you can.<br />
# Press down firmly onto the PDMS to form a seal with the coverslip. <br />
# Repeat with remaining PDMS devices.<br />
# Place sealed devices in a 60°C oven for 5 minutes.<br />
# If possible, leave the devices overnight before use to ensure the best seal.<br />
<br />
==Connect tubing==<br />
<br />
[[Image: ExampleTubing.jpg|thumb|right|350 px|<caption>Inlet and outlet tubing.</caption>]]<br />
<br />
Using a scalpel, cut the following lengths of tubing as marked on their packages. A summary of the tubing needed is below. Cutting the tubing on an angle will make it easier to sleeve together.<br />
{| class="wikitable"<br />
! style="text-align:center;"| Tubing label<br />
! Quantity<br />
! Length to cut (in inches ")<br />
! Description<br />
! Inner diameter (ID, ")<br />
! Outer diameter (OD, ")<br />
|-<br />
| Inlet tubing #1<br />
| 2<br />
| 11"<br />
| Thin tygon tubing, semi flexible<br />
| 0.02<br />
| 0.06<br />
|-<br />
| Inlet tubing #2<br />
| 2<br />
| 10"<br />
| Thick silicone tubing, very flexible<br />
| 1/32<br />
| 3/32<br />
|-<br />
| Long outlet tubing<br />
| 1<br />
| 20"<br />
| Thick tygon tubing, semi flexible<br />
| 1/16<br />
| 1/8<br />
|-<br />
| Short outlet tubing<br />
| 1<br />
| 12"<br />
| Thick tygon tubing, semi flexible<br />
| 1/16<br />
| 1/8<br />
|}<br />
<br />
Collect the following:<br />
# For each inlet, sleeve inlet tubing #1 into inlet tubing #2. Insert a luer-to-barbed fitting into the open end of inlet tubing #2.<br />
# Sleeve a third luer-to-barbed fitting into one end of the long outlet tubing.<br />
# Insert a bent pink needle into each of the PDMS inlet and outlet holes.<br />
# Twist on the barbed fitting of the inlet tubing to the inlet needles, and set the outlet tubing aside.<br />
<br />
Your device is now ready! Keep all your parts stored in a clean petri dish with your microscope, until you are ready to take flow data in Part 3.<br />
<br />
==References==<br />
<references/><br />
<br />
{{Template:Assignment 8 flow channel & two-color microscope navigation}}</div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_5_OverviewAssignment 5 Overview2020-03-23T13:34:47Z<p>Juliesutton: /* Assignment details */</p>
<hr />
<div>[[Category:Lab Manuals]]<br />
[[Category:20.309]]<br />
[[Category:Optical Microscopy Lab]]<br />
{{Template:20.309}}<br />
__NOTOC__<br />
<br />
<br />
==Assignment details ==<br />
This assignment has 2 parts:<br />
<br />
# [[Assignment 5, Part 1: MSD difference tracking and microscope stability|Part 1:]] Implementing difference tracking and measuring the stability of your microscope; <br />
# [[Assignment 5, Part 2: live cell particle tracking of endocytosed beads| Part 2:]] Live cell particle tracking of endocytosed beads.<br />
<br />
Submit your work in on Stellar in a single PDF file with the naming convention <Lastname><Firstname>Assignment5.pdf. <br />
<br />
{{Template:Assignment Turn In|message= Here is a checklist of all things you have to turn in:<br />
For Part 1: <br />
For a slide of fixed beads: <br />
# Plot the MSD versus time interval of <br />
#* the sum trajectories, and<br />
#* the difference trajectories.<br />
<br />
For Part 2:<br />
#Procedure<br />
#*Document the samples you prepared and used and how you captured images (camera settings including frame acquisition rate, number of frames, number of particles in the region of interest, choice of sample plane, etc)<br />
# Data<br />
#* Include a snapshot of the 0.84 &mu;m fluorescent beads monitored.<br />
#* Plot two or more example bead trajectories for each of the samples. (Hint: If you subtract the initial position from each trajectory, then you can plot multiple trajectories on a single set of axes.)<br />
# Analysis and Results<br />
#* Plot the MSD vs time (from the difference trajectories) for untreated and Cyto D treated cells on a single set of log-log axes. Plot the individual MSDs from each pair of beads in each movie. Use a single color for all the untreated cells, and a different color for all the treated cells. Do not include MSDs of particles not endocytosed by the cells.<br />
#* Combine your data with others from the class to increase your sample size.<br />
# Discussion<br />
#* What kind of motion do you see described by your MSD vs &tau; results?<br />
#* What differences do you see between the untreated and Cyto D treated MSD curves? <br />
#* Please suggest an interpretation of the behavior of your cells based on your data.<br />
#* Include a thorough discussion of error sources and your approaches to minimize them. As in Assignment 4, list out the error sources in a table, including a category for the error source, type of error (random, systematic, fundamental, technical, etc.), the magnitude of the error, and a description and way to minimize each one. Your MSD measurements of the still beads (from Part 1 of this assignment) will be useful for estimating the magnitude of several significant error sources.<br />
<br />
Include any MATLAB code you've written as an appendix to your assignment.<br />
}}<br />
<br />
==Code examples and simulations==<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[Matlab: Scalebars]]<br />
* [[Calculating MSD and Diffusion Coefficients]]<br />
<br />
{{Template:Assignment 5 navigation}}<br />
<br />
<br />
<br />
{{Template:20.309 bottom}}</div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_5,_Part_1:_MSD_difference_tracking_and_microscope_stabilityAssignment 5, Part 1: MSD difference tracking and microscope stability2020-03-23T13:33:23Z<p>Juliesutton: /* Develop sum and difference trajectory code */</p>
<hr />
<div>[[Category:Lab Manuals]]<br />
[[Category:20.309]]<br />
[[Category:Optical Microscopy Lab]]<br />
{{Template:20.309}}<br />
<br />
This is Part 1 of [[Assignment 5 Overview| Assignment 5]].<br />
<br />
[[File:How will the plots differ.jpg|center]]<br />
==Overview==<br />
In the last assignment, you made movies of particles as they[https://www.dancetime.com/dance-styles/jitterbug-dance/ jitterbugged] and [https://www.youtube.com/watch?v=oEEY1tq3-ak waltzed] through your microscope's field of view. You developed code to track each particle's unique steps, and you computed each particle's mean squared displacement (MSD), diffusion coefficient, and the implied viscosity of the solutions the particles were suspended in. In this assignment, you'll be tracking particles again. But this time the particles are inside of cells. The cells have been cultured with 0.84 &mu;m fluorescent microspheres, which enter the cells through the process of [https://en.wikipedia.org/wiki/Endocytosis endocytosis]. They are the same kind of cells you took pretty pictures of in assignment 3 (NIH 3T3). Trapped inside of the cells, the particles inside of cells will appear to be stationary to your naked eye. But they are definitely moving &mdash; albeit on a scale much smaller than the particles in glycerin solutions you measured previously.<br />
<br />
How do we know whether our microscope is even capable of measuring such small motions? Could we only be measuring artifacts (like vibrations of the microscope, or drifting of the stage)? <br />
<br />
By measuring the MSD of particles that have been glued down to a slide, we can actually diagnose what types of noise sources are contributing to our measurement, and thus what is the actual smallest motion (not due to noise) that we can measure. Some noise sources (like shot noise) are unavoidable, but others, (like mechanical vibrations and drift) we may be able to correct for.<br />
<br />
==MSD of sum and difference trajectories==<br />
{{:MSD of Sum and Difference Trajectories}}<br />
<br />
As you can see, measuring the MSD of the ''difference'' trajectories for two particles completely cancels out the mechanical noise. We can use the ''sum'' trajectories for two particles to diagnose the amount of mechanical noise initially present in your system: the difference between the MSDs of sum and difference trajectories is equal to (twice) the mechanical noise.<br />
<br />
==Develop sum and difference trajectory code==<br />
<br />
Modify your code from Assignment 4 to be able to track the ''sum'' and ''difference'' trajectories of particles in a movie.<br />
<br />
Steve actually generously shared his code with you!<br />
<pre><br />
function [ DifferenceTrajectories, SumTrajectories ] = CalculateSumAndDifferenceTrajectories( Trajectories )<br />
<br />
numberOfParticles = max( Trajectories(:,4) );<br />
allCombinations = nchoosek( 1:numberOfParticles, 2 );<br />
numberOfCombinations = size( allCombinations, 1 );<br />
<br />
SumTrajectories = cell( numberOfCombinations, 1 );<br />
DifferenceTrajectories = cell( numberOfCombinations, 1 );<br />
<br />
virtualParticleNumber = 1;<br />
<br />
for ii = 1:numberOfCombinations<br />
firstParticleIndex = allCombinations( ii, 1 );<br />
secondParticleIndex = allCombinations( ii, 2 );<br />
firstTrajectory = Trajectories( Trajectories(:, 4) == firstParticleIndex, : );<br />
secondTrajectory = Trajectories( Trajectories(:, 4) == secondParticleIndex, : );<br />
<br />
framesInCommon = intersect( firstTrajectory(:, 3), secondTrajectory(:, 3 ) );<br />
<br />
if( ~isempty( framesInCommon ) )<br />
firstTrajectory = firstTrajectory(ismember(firstTrajectory(:, 3 ), framesInCommon), :);<br />
secondTrajectory = secondTrajectory(ismember(secondTrajectory(:, 3 ), framesInCommon), :);<br />
thisDifference = (firstTrajectory - secondTrajectory)/sqrt(2);<br />
thisDifference(:,3) = framesInCommon';<br />
thisDifference(:,4) = virtualParticleNumber;<br />
DifferenceTrajectories{ii} = thisDifference;<br />
thisSum = (firstTrajectory + secondTrajectory)/sqrt(2);<br />
thisSum(:,3) = framesInCommon';<br />
thisSum(:,4) = virtualParticleNumber;<br />
SumTrajectories{ii} = thisSum;<br />
virtualParticleNumber = virtualParticleNumber + 1;<br />
end<br />
end<br />
<br />
DifferenceTrajectories = vertcat( DifferenceTrajectories{:} );<br />
SumTrajectories = vertcat( SumTrajectories{:} );<br />
<br />
end<br />
</pre><br />
<br />
==Stability of microscope for particle tracking==<br />
[[Image:StabilityPlot2.jpg|right|400px|thumb|Your stability plot should look something like this.]]<br />
Once you have developed and tested your code, use the following procedure to measure the location precision of your microscope: <br />
<br />
# Bring a slide with fixed beads into focus. <br />
#*Choose a field of view in which you can see at least 3 beads using the 40× objective. <br />
#* Limit the field of view to only those beads by choosing a region of interest (ROI) in <tt>UsefulImageAcquisition</tt> tool. (Otherwise, you will have a gigantic movie that is hard to work with.)<br />
# Acquire a movie of beads for 3 minutes at a frame rate of at least 10 fps.<br />
# Use the code you developed to track the particles, and calculate the sum and difference trajectories. <br />
<br />
{{Template:Assignment Turn In|message =Plot the MSD versus time interval of <br />
# the sum trajectories, and<br />
# the difference trajectories.<br />
}}<br />
<br />
The motion of particles endocytosed by 3T3 cells requires an extremely stable microscope, otherwise the real motion of the beads will be obscured by noise. Your microscope will be considered stable enough if the MSD for the ''difference'' trajectory of two fixed particles starts out less than 500 nm<sup>2</sup> and remains less than 1000 nm<sup>2</sup> up to t = 180 s. If the MSD is larger than that, refine your apparatus and methodology until you achieve that goal.<br />
<br />
<br />
<br />
<br />
{{Template:Assignment 5 navigation}}<br />
{{Template:20.309 bottom}}</div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_5:_Spring_2020Assignment 5: Spring 20202020-03-20T20:05:57Z<p>Juliesutton: /* Assignment 5 for Spring 2020 */</p>
<hr />
<div>==Assignment 5 for Spring 2020 ==<br />
<br />
<br />
# Follow the original guidelines for [[Assignment 5, Part 1: MSD difference tracking and microscope stability|Assignment 5, part 1]], using the instructor's movie of 0.84 <math>\mu m</math> beads fixed to a slide. <br />
# For Part 2, complete numbers 2 - 5 of the [[Assignment 5, Part 2: live cell particle tracking of endocytosed beads|original assignment]] <br />
#* You do not need to document your cell-preparation procedure since you didn't get a chance to do it!<br />
#* Analyze the movies of 0.84 <math>\mu m</math> beads endocytosed by NIH 3T3 cells that were recorded instructors. <br />
#* You do not need to share your data with other groups.<br />
<br />
{{Template:Assignment 5 navigation}}</div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_5:_Spring_2020Assignment 5: Spring 20202020-03-20T19:57:50Z<p>Juliesutton: /* Assignment 5 for Spring 2020 */</p>
<hr />
<div>==Assignment 5 for Spring 2020 ==<br />
<br />
<br />
# Follow the original guidelines for [[Assignment 5, Part 1: MSD difference tracking and microscope stability|Assignment 5, part 1]], using the instructor's movie of 0.84 <math>\mu m</math> fixed to a slide. <br />
# For Part 2, complete numbers 2 - 5 of the [[Assignment 5, Part 2: live cell particle tracking of endocytosed beads|original assignment]] <br />
#* You do not need to document your cell-preparation procedure since you didn't get a chance to do it!<br />
#* Analyze the movies of 0.84 <math>\mu m</math> beads endocytosed by NIH 3T3 cells that were recorded instructors. <br />
#* You do not need to share your data with other groups.<br />
<br />
{{Template:Assignment 5 navigation}}</div>Juliesuttonhttp://measurebiology.org/wiki/Assignment_5:_Spring_2020Assignment 5: Spring 20202020-03-20T19:54:57Z<p>Juliesutton: Created page with "==Assignment 5 for Spring 2020 == # Follow the original guidelines for Assignment 5, part 1, using..."</p>
<hr />
<div>==Assignment 5 for Spring 2020 ==<br />
<br />
<br />
# Follow the original guidelines for [[Assignment 5, Part 1: MSD difference tracking and microscope stability|Assignment 5, part 1]], using the instructor's movie of 0.84 <math>\mu m</math> fixed to a slide. <br />
# For Part 2, complete numbers 2 - 5 of the [[Assignment 5, Part 2: live cell particle tracking of endocytosed beads|original assignment]] (you do not need to document your cell-preparation procedure since you didn't get a chance to do it!). Analyze the movies of 0.84 <math>\mu m</math> beads endocytosed by NIH 3T3 cells that were recorded instructors. <br />
<br />
{{Template:Assignment 5 navigation}}</div>Juliesutton