Thisprotocolisveryeffectiveandsimple.ThefactthatthecellsaregrownatlowtemperaturegreatlyincreasetheODrangeduringwhichthecellswillprepupathighefficiency(Inoueetal.,1990). Chillthefollowing:>100mlice-coldTB,centrifuge(GSArotor),500mlcentrifugebottle,andsterilefreezingtubes(Eppendorftubesw/goodcaps) -Innoculate250mlSOBmedia(2literflask)w/10-12coloniesbacteriafromafreshplateShakewellat18°C-RT;growbugstoA600~0.6(ON-2days).AnODof0.6isideal,butanywherefrom0.2to1.0willworkwell.[Alternatively,trydilutinganovernightculturetoOD=0.1andthengrowingatRTfor~6hrs.] -Placeonice10minutes. -Transfertocentrifugebottle;spin2500xg,10min,4°C. -ResUSPendpelletin80mlice-coldTB;placeonicefor10min. -Spinagain2500xg,10min,4°C. -Resuspendin20mlice-coldTB. -AddDMSO,whileswirling,tofinal7%(~1.4ml). -Placeonice,10min.Dispenseintofreezingtubes(~0.5-1mlea) -FreezeinliquidN2;transferto-80°. 1literSOB:20gbactotryptone,5gbacto-yeastextract,0.5gNaCl Dissolveallin950mlddH2O;add10ml250mMKCl;pHto7.0withNaOH Adjustvolto1liter;Autoclave.Justbeforeuse,add5mlsterile2MMgCl2perliter TBperliter 10mMPIPESNasalt3.35g 15mMCaCl2(Fisher)2.2g 250mMKCl(Aldrich)18.64g 55mMMnCl2(Aldrich)10.9g CombinePIPES,CaCl2,KCl;pHto6.7;addMnCl2;filtersterilize REFERENCES Inoue,H.,Nojima,H.,andOkayama,H.(1990).HighefficiencytransformationofEscherichiacoliwithplasmids.Gene96,23-8.