T4连接酶体系

Home›LigationProtocolwithT4DNALigase(M0202)LigationProtocolwithT4DNALigase(M0202)Protocol1.Setupthefollowingreactioninamicrocentrifugetubeonice.(T4DNALigaseshouldbeaddedlast.Notethatthetableshowsaligationusingamolarratioof1:3vectortoinsert.)COMPONENT20μlREACTION10XT4DNALigaseBuffer*2μlVectorDNA(3kb)50ng(0.025pmol)InsertDNA(1kb)50ng(0.076pmol)Nuclease-freewaterto20μlT4DNALigase1μl*TheT4DNALigaseBuffershouldbethawedandresuspendedatroomtemperature.2.Gentlymixthereactionbypipettingupanddownandmicrofugebriefly.3.Forcohesive(sticky)ends,incubateat16°Covernightorroomtemperaturefor10minutes.4.Forbluntendsorsinglebaseoverhangs,incubateat16°Covernightorroomtemperaturefor2hours(alternatively,highconcentrationT4DNALigasecanbeusedina10minuteligation).5.Chilloniceandtransform1-5μlofthereactioninto50μlcompetentcells.