Yeast Prep for FACS
[Adapted from Nash et al. EMBO, 7(13):4335-4346; 1988.]1. Spin down 1E7 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 deg C). Keep samples on rotator.
3. Pellet cells (1 minute) and resuspend in 1 ml of 50 mM Na 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 deg C for 1 hour or overnight at 37 deg C.
6. Pellet and wash cells. Pellet again and resuspend in 1 ml of Na citrate.
7. Add propidium iodide to 16 µg/ml (e.g. add 16 µl of 1 mg/ml PI).
8. Incubate at room temperature for 30 minutes.
9. Proceed with FACS analysis.
[***The resulting sample is usually about 10X too concentrated for analysis on the FHCRC Becton Dickinson machines, so adjust the protocol accordingly or dilute the final sample before analysis.]
Last Modified: Aug 29, 2001