1.Inoculatetheentirecolonyin5mlYPDcultureO.N.or12hours 2.DiluteintoYPAmedium(1%yeastextract,2%Bacto-peptone,2%KOAC)toOD600~0.2,Vigorousshaking. 3.Fortsmutants,~15hoursat23C,others~10hoursat30C,toreachOD1.2-1.4(un-buddedcells~85%.ODisimportant.Donotdilutebackifovershoot.). 4.Washinpre-warmedH2Oonce,thentransfercellstosamevolumeofSPMmedium(2%KOAC).Pachytenecellswillenrichafter~5hourssporulationwithwt. 5.Fortsmutantsandcontrols,pre-incubateat23Cfor1-2hours,thenshiftupto34Corotherdesiredtemperatures.Reference:Cha,etal.,2000,Genes&Dev.14:493-503