NEB　T4连接酶使用说明

介绍: Catalyzestheformationofaphosphodiesterbondbetweenjuxtaposed5'phosphateand3'hydroxylterminiinduplexDNAorRNA.

ThisenzymewilljoinbluntendandcohesiveendterminiaswellasrepairsinglestrandednicksinduplexDNA,RNAorDNA/RNAhybrids.

来源: PurifiedfromE.coliC600pcl857pPLc28lig8(2).

用途: Cloningofrestrictionfragments Joininglinkersandadapterstoblunt-endedDNA

热失活: 65°Cfor10minutes

1X反应缓冲液成分: 50mMTris-HCl 10mMMgCl2 1mMATP 10mMDithiothreitol pH7.5@25°C

酶活定义: Oneunitisdefinedastheamountofenzymerequiredtogive50%ligationofHindIIIfragmentsofλDNA(5′DNAterminiconcentrationof0.12μM,300-μg/ml)inatotalreactionvolumeof20μlin30minutesat16°Cin1XT4DNALigaseReactionBuffer.

储存缓冲液成分: 10mMTris-HCl 50mMKCl 1mMDithiothreitol 0.1mMEDTA 50%Glycerol pH7.4@25°C

保存温度: -20°C

使用须知: 1.ATPisanessentialcofactorforthereaction.ThiscontrastswithE.coliDNAligasewhichrequiresNAD. 2.TodiluteT4DNALigasethatwillsubsequentlybestoredat-20°C,50%glycerolstoragebuffershouldbeused;todiluteforimmediateuse,1XT4DNALigaseReactionBuffercanbeused. 3.LigationcanalsobeperformedinanyofthefourrestrictionendonucleaseNEBuffersorinT4PolynucleotideKinaseBufferiftheyaresupplementedwith1mMATP.

室温连接反应: Forconvenience,ligationsmaybedoneatroomtemperature(20-25°C).Forcohesive(sticky)ends,use1μlofT4DNALigaseina20μlreactionfor10minutes.Forbluntends,use1μlofT4DNALigaseina20μlreactionfor2hoursor1μlhighconcentrationT4DNALigasefor10minutes.

推荐的使用条件: 1XT4DNALigaseReactionBuffer Incubateat16°C.

使用说明书pdf下载

常见问题：

WhataresomepotentialproblemswiththeligationreactionusingT4DNALigasethatcanleadtotransformationfailure?Whataresomeotherproblemsthatshouldbeconsideredwhentroubleshootingatransformationproblem?WhatproblemscanbeencounteredintherestrictiondigestthatcancauseligationusingT4DNALigaseorsubsequenttransformationtofail?WhatcontrolsshouldberuntotestthecellsandDNAwhenusingT4DNALigase?WhenshouldT4DNALigasebetheenzymeofchoice? CantheT4DNALigasebeusedwiththeQuickLigasebuffer?HowmuchDNAshouldbeusedinaligationusingT4DNALigase?CanT4DNALigasebeusedinotherNEBuffers?CanT4DNALigasebeheatinactivated?