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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 g/ml (e.g. add 16 l 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.]

 
Prepared By:
Michael McMurray
(206) 667-6660
Last Modified: Aug 29, 2001


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