Yeast Prep for FACS with Propidium Iodide (Cell-cycle or DNA content analysis)
Adapted from Nash et al. EMBO, 7(13):4335-4346;1988
- Spin down 107 cells in a microfuge tube for 1 minute at 6000g.
- Resuspend pellet in 1mL of 70% EtOH. Fix for at least 60 minutes at room temperature (or up to several days at 4°C). Keep samples on rotator.
- Pellet cells for 1 minute at 6000g and resuspend in 1mL of 50mM Na Citrate pH7.0.
- Sonicate at 30% for 15 seconds, pellet for 1 minute at 6000g and resuspend in 1mL of Na citrate.
- Add RNAse A to 0.25mg/mL final concentration. Incubate at 50°C for 1 hour or overnight at 37°C.
- Pellet for 1 minute at 6000g and wash cells. Pellet again and resuspend in 1mL of Na citrate.
- Add Propidium Iodide to 16ug/mL (e.g add 16uL of 1mg/mL PI).
- Incubate at room temperature for 30 minutes.
- Proceed with FACS analysis.