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        NEB T4连接酶使用说明

        互联网

        39246

        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?

        (责任编辑:大汉昆仑王)

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