SteveHahn LastModifiedDecember1997 Thismethodworkswellwhentransformingwithaplasmidandisrapid.(forhighestefficiencytransformation,seeDMSOtransformationmethod) Grow100mlcellsovernightinYPDtoanA600of~1.0-2.0.(Asmallblobofcellsfromaplateinoculatedinto100mlYPDandgrownfor14hrat30oisusuallyfine).Harvestcells(100ml)bycentrifugation(5Krpmfor5mininGSA).Ifcelldensityisgreaterthan1.0,usecorrespondinglylesscells.ResUSPendcellsin5mlTEpH7.5andtransferto15mlscrewcaptube.Spin2mininclinicalcentrifuge.Resuspendcellpelletin5mlTE+0.1Mlithiumacetate.Spin2mininclinicalcentrifuge.Resuspendcellpelletin2mlTE+0.1Mlithiumacetate.Incubateontuberollerat30ofor1hr(canbeincubatedforuptofourhourswithnoilleffects).BoilhighmolecularweightsalmonspermDNAfor3-5min.Rapidlychillonice(thisseparatesDNAstrands,makingitsinglestranded). InasterileEppendorftube,add:1.10microliters(10mg/ml)singlestrandedhighmolecularweightsalmonspermDNA2.PlasmidDNAfortransformation(1microgramshouldgive>1000transformants;1-2microlitersofmini-prepDNAworkswell).3.0.2mlcellstreatedwithLiOAcfromabove.4.1ml40%PEG4000,1XTEpH7.5,0.1MLithiumacetate.Mixbyvortexingandincubateat30degreesfor30min.Heatshockcellsfor15min.at42degrees.Spin5secinmicrofugeandremovesupernatantbydumpingoff.Resuspendcellpelletin1mlTEusingasterilepipetmantipandvortexing.Spininmicrofuge5sec.Removesupernatantbydumpingoff.Resuspendcellpelletin0.5mlTEbyvortexing.Plate0.2mltoselectiveplates.