E.colicellscanbedisruptedinanalkalinesolutioncontainingdetergent.ThelysatecontainsenoughDNAtobedetectedinasinglelaneofanagarosegelprovidedthattheplasmidhavepUC-derivedreplicationorigin.Thefollowingprotocolismodifiedfromthatdescribedinthefirstversionof"MolecularCloning"(T.Maniatis,E.F.FrischandJ.Sambrook,ColdSpringHarborLaboratoryPress,1982).(NOTE:Iseldomusethisprocedurenow,sincePCR-basedproceduredescribedhereismorereliable.) 1.Growbacterialcoloniestoalargesize(2-3mm)onanagarmediumcontininganappropriateantibiotic. 2.Usingasteriletoothpick,transferasmallquantityofthecolonytoamasterplate.Transfertheremainderofthecolonytoamicrofugetubecontaining20microlitersof50mMNaOH,0.5%SDS,5mMEDTA(crackingbuffer). 3.Incubatethetubeat55Cfor30min. 4.Vortexvigorouslyfor1min.* 5.AddanappropriateamountofloADIngbuffer**.Loadthecontentsontoanagarosegelwithoutethidiumbromide.Asacontrol,loadtheplasmidvectorwithoutinsertononelane. 6.Afterelectrophoresis,stainthegelbysoakingfor30minutesinasolutionofethidiumbromide(0.5microgram/mlineitherwaterorelectrophoresisbuffer). 7.UnderUV-Illuminator,plasmidDNAshouldbevisIBLebetweenE.coligenomicDNA(20-30kb)andlowmolecularweightRNAs. *Atthisstep,longgenomicDNAiscutintosmallerpiecesofabout20-30kb.Althoughtheoriginalprotocolin"MolecularCloning"doesnotcontainthisstep,vigorousvortexingisnecessarysincelonggenomicDNAinthelysateistroublesomeinloadingthesampleontotheagarosegel.**Addtheloadingbufferjustbeforeelectrophoresis,sincebromophenolblueisrapidlydegradedinthealkalinesolution.ColonyCracking:QuickTestforInsertsinPlasmids