YeastPrepforFACS
[AdaptedfromNashetal.EMBO,7(13):4335-4346;1988.]
1.Spindown1E7cellsinamicrofugetubefor1minute.2.ResUSPendpelletin1mlof70%EtOH.Fixforatleast60minutesatroomtemperature(oruptoseveraldaysat4degC).Keepsamplesonrotator.3.Pelletcells(1minute)andresuspendin1mlof50mMNacitratepH7.0.4.Sonicate(30%for15sec),pellet,andresuspendin1mlofsamesolution.5.AddRNaseAto0.25mg/ml.Incubateat50degCfor1hourorovernightat37degC.6.Pelletandwashcells.Pelletagainandresuspendin1mlofNacitrate.7.Addpropidiumiodideto16µg/ml(e.g.add16µlof1mg/mlPI).8.Incubateatroomtemperaturefor30minutes.9.ProceedwithFACSanalysis.[***Theresultingsampleisusuallyabout10XtooconcentratedforanalysisontheFHCRCBectonDickinsonmachines,soadjusttheprotocolaccordinglyordilutethefinalsamplebeforeanalysis.]
