请使用支持JavaScript的浏览器! DNA水解酶与DNA限制性内切酶、DNA酶的区别概述_实验方法__蚂蚁淘,【正品极速】生物医学科研用品轻松购|ebiomall -蚂蚁淘商城
当前位置: > 首页 > 技术文章 >
DNA水解酶与DNA限制性内切酶、DNA酶的区别概述_实验方法_
来自 : 蚂蚁淘

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.

免责声明 本文仅代表作者个人观点,与本网无关。其创作性以及文中陈述文字和内容未经本站证实,对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺,请读者仅作参考,并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的,应该授权范围内使用,并注明“来源:蚂蚁淘”。违反上述声明者,本网将追究其相关法律责任。
相关文章