Two-color Yeast Protocols

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Reagents

All part numbers are from Millipore-Sigma unless stated otherwise

YPD Broth and plates

  • YPD Broth, Y1375-250G
  • Agar

Synthetic Complete (SC) medium

  • LoFlo Yeast nitrogen base without amino acids and without folic acid and without riboflavin, CYN6502 from formedium.com
  • Yeast synthetic dropout powder, Y1751-20G
  • Histidine, H8000-5G
  • Glucose
  • NaCl

Concanavalin A

  • Concanavalin A type IV, C2010-100MG
  • PBS

Protocols

Concanavalin A

Make 250 uL aliquots of 1mg/ml in PBS.

  1. Dissolve 5 mg ConA in 5 mL PBS.
  2. Aliquot into 20x 250 uL.
  3. Store at -20C.


YPD broth

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.

  1. Mix 12.5g YPD broth powder (from sigma) with 250 mL water.
  2. Autoclave for 25 minutes at 121 C

YPD plates

  1. For 10 plates, mix
    • 12.5g YPD broth powder
    • 3.75g agarose
    • 250 mL water.
  2. Autoclave for 25 minutes at 121 C
  3. When cool enough to handle, pour ~25mL of YPD-agar into each plate

50% Glucose Stock Solution

Glucose MW = 180.16 g/mol

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...

For a 200ml stock solution:

  1. Measure 200ml H2O and pour it into a large bottle that will be used to store stock solution.
  2. Add a stir bar to the bottle and mark the water level with a piece of tape.
  3. Pour out 1/2 of the water, leaving the stir bar behind.
  4. Add 100g glucose to the bottle while stirring to dissolve.
  5. When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.
  6. When all (or most) of the glucose is dissolved, top up with water to the 200mL mark.

1M NaCl Stock solution

NaCl MW = 58.44g/mol

  • Dissolve 29.22 g NaCl in 500mL H2O

Synthetic Complete culture media

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.

I like to make a batch of 2x stock SC medium, then divide it into three bottles:

  1. "low salt" medium, which is the same as regular SC medium, to be used for flow experiments
  2. "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments
  3. the remaining SC medium I use for yeast culture.

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.

For 750 mL of 2 x low fluorescence SC stock

  1. In a 1L bottle, mix the following:
    • 2.88 g Drop out powder (-His),
    • 114 mg histidine,
    • 10.05 g LoFlo nitrogen base
    • 750 mL water

Don't forget to add glucose (see below) for SC growth medium!

For 500 mL each of low and high salt medium

  1. Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.
  2. Repeat step 1 for a second bottle and label it "low salt" medium.
  3. For the low salt medium,
    • Measure out 100 mL of DI water and pour into bottle.
  4. For high salt medium,
    • Measure out 100 mL of 1M NaCl and pour into bottle.
  5. Top up each bottle with 130 mL of water and add 20 ml of 50% glucose.
  6. Filter sterilize using a 0.2um filtration system into sterile bottles.

Growing yeast cultures

Plate

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.

20.309 freezer stock.

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.

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.

Making new freezer stock

To make a new freezer stock,

  1. grow up cells on YPD plate,
  2. scrape up a bunch of healthy colonies with with sterile toothpick,
  3. mix toothpick into cryo tube with YPD+30% glycerol

Note Jan 2021: I have yet to do this, but it will need to be done eventually.

Liquid Culture

(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.)

The cells will recover more quickly if the YPD plate is at room temperature, but you can inoculate from the fridge if necessary. 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.

  1. Add 10 mL of SC medium to a 125mL flask.
  2. Scrape up a small amount of culture from YPD plate using a sterile stick or inoculation loop.
  3. 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.
  4. Attach the flask to the orbital shaker and shake at 180 rpm overnight.
    • Since we are doing the experiments at room temperature, it's best to also grow the yeast at room temperature.
    • 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.
    • 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.

The day before the experiment:

  1. 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)
  2. At about 9am, dilute the culture to get OD600 = 0.4 at 1pm. Call this Culture 1.
  3. Around 11am, dilute the overnight culture once to get 0.4 at 3:30 pm, and 0.4 at 6pm.
  4. At 7pm, dilute saturated culture for next day to repeat.


Target yeast concentrations
Time Culture 1 Culture 2 Culture 3
1 pm 0.4
3:30 pm 0.8 0.4
6 pm 0.8 0.4
8:30 pm 0.8

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.

Fall 2022 note: Here are two dilution scenarios that have worked perfectly.

  1. OD600 = 0.25 at 12pm on 11/26 ==> aim to dilute yeast for OD600 = 0.4 at 12pm on 11/27
    • 24 hours ~ 10 x 2.5 h (our yeast doubles every 2.5 h)
    • There will be 2^10 = 1024 more yeast in 24 hours
    • Today we want to prepare a dilution so that OD600 = 0.4 / 2^10 = 4 x 10^-4
    • Since our starting OD600 is 0.25, we need to dilute it 625 times in order to reach OD600 = 4 x 10^-4
    • Mix 10 mL SC medium with 20 uL of today's yeast
  1. OD600 = 0.55 at 4pm on 11/27 ==> aim to dilute yeast for OD600 = 0.4 at 12pm on 11/28
    • 20 hours ~ 8 x 2.5 h
    • There will be 2^8 =256 more yeast in 20 hours
    • Today we want to prepare a dilution so that OD600 = 0.4 / 2^8 = 1.5 x 10^-3
    • Since our starting OD600 is 0.55, we need to dilute it 352 times in order to reach OD600 = 1.5 x 10^-3
    • Mix 10 mL SC medium with 30 uL of today's yeast