Yeast FACS Analysis

1. Spin down 2E7 cells in a microfuge tube for 1 minute.
   
   
2. Resuspend pellet in 1 ml of 70% EtOH. Fix for at least 60 minutes at room temperature (or up to several days at 4^oC). Keep samples on rotator.
   
   
3. Pellet cells (1 minute) and resuspend in 1 ml of 50 mM sodium citrate pH 7.0.
   
   
4. Sonicate (30% for 15 sec), pellet, and resuspend in 1 ml of same solution.
   
   
5. Add RNase A to 0.25 mg/ml. Incubate at 50^oC for 1 hour or overnight at 37^oC.
   
   
6. Pellet and wash cells. Pellet again and resuspend in 1 ml of sodium citrate.
   
   
7. Add propidium iodide to 16 ug/ml (e.g. add 16 ul of 1 mg/ml PI).
   
   
8. Incubate at room temperature for 30 minutes.
   
   
9. Proceed with FACS analysis.
   
   ***The resulting sample is usually about 20X too concentrated for analysis on the Becton Dickinson machines, so adjust the protocol accordingly or dilute the final sample before analysis.