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.

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