Difference between revisions of "Two-color Yeast Protocols"

Revision as of 18:48, 14 August 2019

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

• 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.

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:

1. Measure 200ml H2O and pour it into a bottle large 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

SC media

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 SC stock

1. 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

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.
6. Autoclave both bottles, as well as 50% glucose stock for 25 minutes.
7. Use a sterile pipette to add 20 ml of 50% glucose to each bottle.

YPD broth

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
   • 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