ATCG

Yeast Prep for FACS with Propidium Iodide (Cell-cycle or DNA content analysis)

Adapted from Nash et al. EMBO, 7(13):4335-4346;1988

  1. Spin down 107 cells in a microfuge tube for 1 minute at 6000g.
  2. 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.
  3. Pellet cells for 1 minute at 6000g and resuspend in 1mL of 50mM Na Citrate pH7.0.
  4. Sonicate at 30% for 15 seconds, pellet for 1 minute at 6000g and resuspend in 1mL of Na citrate.
  5. Add RNAse A to 0.25mg/mL final concentration. Incubate at 50°C for 1 hour or overnight at 37°C.
  6. Pellet for 1 minute at 6000g and wash cells. Pellet again and resuspend in 1mL of Na citrate.
  7. Add Propidium Iodide to 16ug/mL (e.g add 16uL of 1mg/mL PI).
  8. Incubate at room temperature for 30 minutes.
  9. Proceed with FACS analysis.