LindaHoskins,HahnLab

Lastmodified10/16/98

1.Growa50mlO/NYPDyeastcultureat30degreesC.2.Determinecelldensityandtransferanamountofculturecorrespondingto1x108cellstoseveral15mlconicaltubes.Spindowncellsandwashwith5mlsterilewater.Spindowncellsandwashwith5ml0.1Msodiumphosphate(NaH2PO4)buffer,pH7.0.SpindowncellsandresUSPendin1.7mlbuffer.3.Transfercellstoglassculturetubes.Inhood,add50ulEMS(ethylmethanesulfonate)(alsohaveonecontroltubewithoutEMS).Incubateonrollerat30degreesforvaryingamountsoftime,between20min.andonehour.4.Ateachtimepoint,add8mlsterile5%sodiumthiosulfate(autoclaved).ThiswillstopthemutagenesisbyinactivatingtheEMS.Eachcellsuspensionshouldcontain1x107cells/ml.Saveanaliquotofcellsatthispointtouseinfiguringoutthecellsurvival.*Allglassware,plasticware,andsolutionsthatcomesincontactwithEMSshouldberinsedwith5%sodiumthiosulfatetoinactivatetheEMS.5.Spindowncellsin15mlconicaltubesandresuspendin9mlsterilewater.FigureoutactualcelltiterafterwashbyplatingoutvaryingdilutionsontoYPDplatesat25degreesC.Storecellsat4degreesC.6.Todeterminethecellsurvival,useanaliquotofcellsfromstep4,dilute,andplateontoYPDplatestogiveabout500cells/plate.Incubateat30degreesC.CountthenumberofcoloniesandcompareeachEMStimepointtothenonmutagenizedcells.Usethetimepointthatgivesacellsurvivalof60-70%.7.UsestoredcellstoplateoutvaryingnumbersofcellsontoYPDplates.Alsoplateoutnonmutagenizedcellsasacontrol.(Optional:brieflysonicatecells,~10sec,tomakesurethattherearenoclumpsofcellspresentbeforeplating).Ifsearchingforsuppressorsofatsstrain,incubateoneplateat25degreesCandoneplate37degreesC,lookforcoloniesthatgrowbetteronthe37degreesCplates.Ifsearchingfortsstrains,incubateallplatesat25degreesCuntilcoloniescomeup,replica-platetotwonewYPDplates,incubateoneplateat25degreesCandoneplateat37degreesC,lookfortscoloniesonthe37degreesCplates.