Difference between revisions of "Two-color Yeast Protocols"
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* Dissolve 29.22 g NaCl in 500mL H2O | * Dissolve 29.22 g NaCl in 500mL H2O | ||
− | === | + | === Synthetic Complete culture media === |
I like to make a batch of 2x stock SC medium, then divide it into three bottles: | I like to make a batch of 2x stock SC medium, then divide it into three bottles: |
Revision as of 18:49, 14 August 2019
Contents
Reagents needed
All part numbers are from Millipore-Sigma unless stated otherwise
YPD Broth and plates
- YPD Broth, Y1375-250G
- Agar
Synthetic Complete (SC) medium
- Yeast nitrogen base without amino acids, Y0626-250G
- Yeast synthetic dropout powder, Y1751-20G
- Histidine, H8000-5G
- Glucose
- NaCl
- Concanavalin A type IV, C2010-100MG
- PBS
Protocols
Make 250 uL aliquots of 1mg/ml in PBS.
- Dissolve 5 mg ConA in 5 mL PBS.
- Aliquot into 20x 250 uL.
- Store at -20C.
50% Glucose Stock Solution
Glucose MW = 180.16 g/mol
Each 1L of SC medium requires 40 ml of 50% glucose. For a 200mM stock solution:
- Measure 200ml H2O and pour it into a bottle large that will be used to store stock solution.
- Add a stir bar to the bottle and mark the water level with a piece of tape.
- Pour out 1/2 of the water, leaving the stir bar behind.
- Add 100g glucose to the bottle while stirring to dissolve.
- When all the glucose is added, add more water to 3/4 of the way up to the 200mL mark.
- 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
I like to make a batch of 2x stock SC medium, then divide it into three bottles:
- "low salt" medium, which is the same as regular SC medium, to be used for flow experiments
- "high salt" medium, is regular SC medium supplemented with 200mM NaCl, to be used for flow experiments
- 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 SC stock
- In a 1L bottle, mix the following:
- 2.88 g Drop out powder (-His),
- 114 mg histidine,
- 10.05 g nitrogen base
- 750 mL water
For 500 mL each of low and high salt medium
- Measure 250 ml of 2x SC medium using a 500mL cylinder and add to a 500mL bottle. Label this as the "high salt" medium.
- Repeat step 1 for a second bottle and label it "low salt" medium.
- For the low salt medium,
- Measure out 100 mL of DI water and pour into bottle.
- For high salt medium,
- Measure out 100 mL of 1M NaCl and pour into bottle.
- Top up each bottle with 130 mL of water.
- Autoclave both bottles, as well as 50% glucose stock for 25 minutes.
- Use a sterile pipette to add 20 ml of 50% glucose to each bottle.
YPD broth
- Mix 12.5g YPD broth powder (from sigma) with 250 mL water.
- Autoclave for 25 minutes at 121 C
YPD plates
- For 10 plates, mix
- 12.5g YPD broth
- 3.75g agarose
- 250 mL water.
- Autoclave for 25 minutes at 121 C
- When cool enough to handle, pour ~25mL of YPD-agar into each plate