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.