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 4oC). 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 50oC for 1 hour or overnight at 37oC.

  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.