CohesiveEndLigation

1)Theligationmixturecontainsthefollowing:

1. vectorDNA(~100ng)
2. insertDNA(equimolaror2or3Xmolarconcentrationofvector)
3. wateraddedto18µl

2)Heatthemixtureat45°Cfor5min.tomeltanyannealedcohesiveends.

3)Coolonice.Add

1. ligasebuffer(+ATP)2µl
2. ligase0.5µl

4)Incubatereactionmixtureat14°Cfor2-4hoursorovernightat4°C.

5)Heatligationmixtureat65°Cfor10minutestodeactivatetheligase.

6)Use5-10µloftheligationmixturetotransformcompetentcells.

Note:

a)InorderfortheDNAtocircularise,itisimportantnottousetoohighaconcentrationofDNA(highconcentrationofDNApromotesintermolecularreactionandthereforeformslonglinearmolecules).ConcentrationofvectorDNAshouldnormallybe<10ng/ul.TrydilutingtheDNA5Xandthenfurtherdilutethis5Xtogive3reactionmixturesatdifferentDNAconcentration.Calculationtoobtainthebestvectorandinsertconcentrationisnormallynotrequired.b)Doacontrolligationofthevectorwithoutanyinsert.Ifthebackgroundislow,thereisnoneedforscreeningofthetransformedcellsforvectorwithligatedinsert.c)Forbluntendligation(whichismuchlessefficientcomparetocohesiveendligation),useahigherconcentrationofDNAandligase(~10X).Higherconcentrationofligase(~5x)isalsousefulforstickyendligationifligationtimeisshort.d)Onlyusephosphatasewhenstrictlynecessary.Fewerstepsmeanslessproblem.Ifitisnecessarytouseit,chooseSAP(shrimpalkalinephosphatase-easiertodeactivate)insteadofCIP.OnlyusetherecommendedamountastoomuchcandamagetheendsofDNA(thisisveryimportant-damagedendswon"tligate).e)AvoidusingTBEwhengel-purifyingDNA(useTAEinstead).Alsousethebestgradeagaroseorlowm.p.agaroseforthegel.Contaminants(suchassulphatedagarose)canaffectyourligationefficiency.DNAshouldalwaysbegel-purifiedbeforeligation.f)SomeenzymeshavebeensuggestedtobeabletosticktotheendsofDNAtherebyaffectingligation.Thereisuncertaintyifthisisthecase,orifitisaproblemofenzymecarryingoverduringpurification.InsuchcasestheDNAsolutionneedstobetreatedwithproteinaseKfollowedbyphenol/chloroform.Taqpolymerase,forexample,maynotbecompletelyremovedduringpurificationandcanfillinthedigestedendofDNAandtherebypreventingligation.RestrictionenzymescanalsodigestligatedDNA.Gelpurification,however,shouldremovetherestrictionenzymeswellg)Keepthebufferinsmallaliquotsasthefreezing/defrostingcyclesmaydamagetheATP.AlsodonotusetoohighaconcentrationofATP(max.0.5mM)forblunt-endligationaspolyadenationofblunt-endedDNAmayoccur?h)TheligaseworksbestathighertemperaturebutthereactiontemperatureiskeptlowtoallowtheendsoftheDNAtoannealtogether.TheTmoftheendsaregenerally~12-16°C,butEcoRImaybelower(5-6°C)sinceendsareAATT.(Forblunt-endligation,thetemperaturemaynotbesoimportant,ligationathighertemperaturemaythereforebemoredesirable.)i)Avoidusecondensingagentslikehexaminecobaltchlorideespeciallyforstickyendligation.Theypromoteintermolecularreactionthereforeformconcatamersratherthanclosedplasmids.However,ifyoumustusethem,makesurethere"sK+inthebufferwhichpromotecircularisationofplasmid.j)Oneusefulideainblunt-endligation,addrestrictionenzymesintheligationmixture-normallyblunt-endligationofinserttovectordoesnotreformtherestrictionsite(thereforewhenyoudesigntheoligothatmakesureitdoesn"treformtherestrictionsitewhenligated),thereforeonlyreligatedvectorisdigested.Seeone-tubePCRcloningforanexample.k)DoremembertopolishofftheendsafterPCRifyou"redoingblunt-endligationofPCRproductproducedbyTaqpolymerase.However,there"snoadenylationforotherpolymeraseslikeTli.l)Useonlyhighlycompetentcellsfortransformationincloningwork.Expect50-1000transformants(ifthingsgowellthatis).m)ItispossIBLetoclonegenewithoutdoingligation.Basically,youaddextra11-12basesLICsite(ligationindependentcloningsite).ThePCRproductsaredigestedinthepresenceofT4DNApolymerasewithdTTPordATPwhichexposetheLICsitewhencanannealedwithanotherLICsiteonthevector(availablefromPharmingenandNovagen)whichcanthenbetransformed.SeeHaunetal,Biotechniques13(4)515-8,1992,Rashtchian,CurrentOpinioninBiotechnology6:30-36,1995n)Make-your-ownTA-cloningvector-canuseplasmidpDK101[ATCC77406],aderivativeofplasmidpGEM[R]5fZ(+).DigestwiththeendonucleaseXcmIwhichgivetheoverhangsforcloning.AnotherwayistodigestpBluescriptwithEcoRVandperformaTaqpolymerase3"-terminaladditioninthepresenceofdTTP.SeeTIBSarticle"CloningPCRproductsusingTAvectors"formorediscussiononTA-cloningandalsootherprotocolssitestomakingTvector.

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