SimpleandEfficientMethod(SEM)toMakeCompetentCellsPreparationofFrozenCompetentofDH5α1)250mlTBsolution10mMPipesor10mMHepes,0.65g15mMCaCl2,0.55g250mMKCl,4.66g*55mMMnCl2,2.47gAllthecomponentsexceptforMnCl2weremixedandthepHwasadjustedto6.7with0.1NHCl(Ifyouovershoot,donotreadjustthepHbyaddingbase,instead,discardthesolutionandbeginagain).Then2.475gMnCl2wasaddedtothesolutionanddissolved.Thesolutionwassterilizedbyfiltrationthroughaprerinsed0.45umfilterunit.Dispensethesolutioninto50-mlaliquotsandstoredat4°C.2)OtherSolution:A.100mlof250mMKClsolutionwithddH2O,non-autoclaved:1.86gofKClin100mlddH2O.B.10mlof1MMgCl2+1MMgSO4withddH2O,autoclave.(Prepare1MMgCl2first,thenaddMgSO4intoit)C.50mlof1MglucosewithddH2O,filter,aliquotin1.5ml,storeat-20°CD.For100mlSOBmedium:（Amount/volume）Bactotryptone:2gBactoyeastextract0.5gNaCl0.05gShakeuntilthesoluteshavedissolved.Add10mlofa250mMsolutionofKClAdjustthepHto7.0with5NNaOH(~0.2ml)Adjustthevolumeofthesolutionto100mlwithddH2OSterilizebyautoclavingfor20minat15lb/sq.inonliquidcycleJustbeforeuse,combinethemediumwith1mlof2MMg2+(20mMfinal)E.SOCmedium:SOCmediumisidenticaltoSOBmedium,exceptthatitcontains20mMGlucose.3)Aautoclaved500mlflask.Method:Doitonice!!!1.Usingaautoclavedyellowtip,streakDH5αdirectlyfromafrozenstockinto2mlSOB,shakeO/Nat37°C.2.1mlculturewasinoculatedin100ml(1:100dilution)SOB.Incubateat37°Cwithmoderateagitationfor2~2.5hours,thecelldensityis4~7×107viablecells/mlorOD600=0.5.Tomonitorthegrowthoftheculture,determinetheOD600every20~30min.Or:1.Usingasterileyellowtip,streakDH5αdirectlyfromafrozenstockontothesurfaceofanSOBagarplate.(Don’tthawthefrozenstockofbacteria,sothesingletubeoffrozenstockcanthereforebeusedmanytimes.)Incubatetheplatefor16hoursat37°C.2.Transferfourorfivewell-isolatedcoloniesinto1mlofSOB.Thecoloniesshouldbe1-2mmindiameter.Dispersethebacteriabyvortexingatmoderatedspeed,andthendilutetheculturein100mlofSOBina500mlflask.3.Collectthecultureinto2of50mlFalcontubesandchillonicefor10-15min.4.Pelletthecellsbycentrifugationat750-1000g(2000-3000rpmnaclinicalcentrifuge)for12minat4ºC.Drainthepelletedcellsthoroughlybyinvertingthetubesonpapertowels,andrappingtoremoveanyliquid.AmicroPipettecanbeusedtodrawoffrecalcitrantdrops.5.ResUSPendeachofthecellpelletinTBbypipettingupanddownverygentlyin1mlofTB.Afterthecellpelletwaspipettedtosinglecell,another9mlofTBwasadded.Combinethecellsinthe2tubes.Incubatethecellsonincfor15min.6.Collectthecellsbycentrifugationat750-1000gfor10minat4°C.7.ResuspendeachofthecellpelletinTBbypipettingupanddownverygentlyin1mlofTB.Afterthecellpelletwaspipettedtosinglecell,another7mlofTBwasadded.(to1/12.5oftheoriginalvolume)8.Add280µlofDMSOper8mlresuspendedcells.Mixgentlybyswirling,andstorethesuspensiononicefor15min.9.Addanother280µlofDMSOper8mlresuspendedcells.Mixgentlybyswirling,andthenreturnthesuspensionstoanicebath.10.Distributealiquots100µlwithchilled,sterilepipettetipintochilled1.5mlmicrocentrifugetubes.11.Flashfreezeinliquidnitrogen,thenplaceat-70°C.如果用来建库或者转化比较珍贵的材料可以试一下这种感受态！效率非常高！

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试剂库>>克隆与表达>>转化

试剂库>>细胞培养>>培养基/琼脂混合物

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试剂库>>生物分子>>抗生素/抗真菌素仪器库>>实验室常用设备>>消毒/灭菌

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耗材库>>常用耗材>>手套

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