Yeast FACS Analysis
Spin down 2E7 cells in a microfuge tube for 1 minute.
Resuspend pellet in 1 ml of 70% EtOH. Fix for at least 60 minutes at room temperature (or up to several days at 4oC). Keep samples on rotator.
Pellet cells (1 minute) and resuspend in 1 ml of 50 mM sodium citrate pH 7.0.
Sonicate (30% for 15 sec), pellet, and resuspend in 1 ml of same solution.
Add RNase A to 0.25 mg/ml. Incubate at 50oC for 1 hour or overnight at 37oC.
Pellet and wash cells. Pellet again and resuspend in 1 ml of sodium citrate.
Add propidium iodide to 16 ug/ml (e.g. add 16 ul of 1 mg/ml PI).
Incubate at room temperature for 30 minutes.
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.