LiOAcYEASTTRANSFORMATIONa (seenotesatend) UseSTERILETECHNIQUEthroughout. 1.Inoculatea10mlovernightculturewithasingleyeastcolony.Ifstartingwithayeaststrainwithnoplasmids,useYPD.Ifstartingwithastrainthatalreadyhasoneormoreplasmidsuseliquiddrop-outmedialackingtheappropriatenutrient.bIncubateat30oCinrollerdrumattopspeedovernight.b 2.MeasuretheOD600oftheovernightculture.cMakeanappropriatedilutionoftheovernightcultureinto50mlofmediainasterile250mlflasksothatthefinalOD600is0.2. 3.Incubateat30oC,shakingat200rpm,untilOD600=1.0 4.Transferculture(usingsteriletechnique!)toasterile50mlpolypropylenetube(Oakridgetube).Spininclinicalcentrifugeat3000rpm,2min.Thisandallfollowingmanipulationsaredoneatroomtemperature. 5.Dumpoffmedia(quicklyandcarefully)andadd10mlsterilewater.ResUSPendcellsbyswirlingorgentlevortexing. 6.Pelletcellsinclinicalcentrifugeat3000rpm,2min.Pouroffwater. 7.Add5mlofLiOAc/TEsolution.Resuspendcellsbyswirlingorgentlevortexing. 8.Pelletcellsinclinicalcentrifugeat3000rpm,2min.PouroffLiOAc/TE. 9.Add0.25mlofLiOAc/TEsolution.Resuspendcellsbyswirlingorgentlevortexing. 10.Foreachtransformationaliquot50µlofcellstoasterileEppendorftube.ALWAYSsetupacontroltubethatwillgetnoDNA. 11.AddplasmidDNAd.[Forhigherefficiency(e.g.forlibrarytransformationse)addDMSOto10%(e.g.~6µl)].Mixgently.Add300µl40%PEGinLiOAc/TE.Mixbyinvertingtwoorthreetimes. 12.Donotvortex.InthepresenceofPEGyeastcellsaremorefragile. 13.Incubateat30oCfor30min.Takethistimetocheckthetemperatureofthe42oC,andwarmandlabelplates.Removecellstoroomtemperature. 14.Incubateat42oCforexactly15min.Removetoroomtemperature. 15.Plate200-300µl,includingthebulkofthecells,ontoappropriateplates.Incubateat30oCwithplatesrightsideup.Turnplatesoverafter1day. NOTES aThismethodisbasedontheprocedureofGeitzetal.,1992NucleicAcidsResearch20:1425. SeealsoGeitzLabYeastTransformationHomePage bWhenusingYPD,12to16hoursissufficientfora10mlovernightculture.Growthindrop-outmediamaytakemuchlongersothe"overnight"culturemaybestartedinthemorningforthefollowingday.Alternatively,a50mlovernightculturemaybestarted-whichmayormaynotneedtobedilutedthenextday. cSpectrophotometermeasurementsareonlylinearatOD600between~0.05and1.0.IftheOD600ofthecultureisover1.0itshouldbediluted10-foldtotakethemeasurement.Note:IdeallyoneshouldtrytransformingyeastatdifferentOD600from0.7to1.5toseewhichisbest.Spectscandiffer,andthisapproachiseasierthancountingcells dUsenomorethan1µgplasmidDNA/50µlofcells.Forroutinetransformations,5-10µlofminiprepDNAor0.1-0.5µgmaxiprepDNAissufficient.Forlibrarytransformationsuse1µgplasmidPLUS30µgcarrierDNA(suchasBoehringerMBgradefishspermDNACat#1467140;seealsoSchiestlandGietz(1989)CurrentGenetics16:339-346). eToperform10ormoretransfromations,scaleupbyusinga200mlcultureatstep2,andresuspendin1mlLiOAc/TEsolutionatstep9. SOLUTIONS LiOAc/TE For50ml,inasterile50mlFalcontube. 44.4mlsterilewater 5.0mlsterile1MLiOAc,pH7.0-7.4 0.5mlsterile1MTrisHCl,pH7.5 0.1mlsterile500mMEDTA 40%PEGinLiOAc/TE For50ml,inasterile50mlFalcontube. 40mlsterile50%PEG4000 5.0mlsterile1MLiOAc,pH7.0-7.4 0.5mlsterile1MTrisHCl,pH7.5 0.1mlsterile500mMEDTA 4.4mlsterilewater.