Please find the following protocol:

Preparation of Yeast Cells for FACS DNA Quantitation


Procedure:

1. Harvest 1 to 5 ml of a log-phase yeast culture (5 million cells/ml).

2. Pellet the cells by centrifugation at 1500 X g, remove the supernatant and wash with 1 ml 50 mM Tris, pH 7.5, transfer the cells to a microcentrifuge tube.

3. Pellet the cells again and resuspend in 300 μl 50 mM Tris, pH 7.5 and add 700 μl 100% Ethanol.

4. Incubate the cells 1 hr at room temperature.

5. Pellet the cells again and wash twice in 50 mM Tris, pH 7.5.

6. Pellet the cells, remove the supernatant and resuspend the pellet in 500 μl of 1 mg/ml RNase (boiled) in 50 mM Tris, pH 7.5. Incubate either 1 hr at 37°C and overnight at 4°C or at 37°C for 4 hr.

7. Pellet cells and resuspend in 200 μl of PI Stain.

8. Incubate for 1 hr at room temperature. The samples can be stored at 4°C at this point.

9. Dilute samples 1:10 to 1:50 into PBS, sonicate and perform FACS analysis


Solutions:

PI stain
50 μg/ml Propidium Iodide in PBS (CAUTION Biohazard!)

50 mM Tris, pH 7.5

PBS
pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl


Bioreagents and Chemicals:

Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Ethanol
Potassium Phosphate, Monobasic
Tris
Propidium Iodide
RNase


Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p875