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)
