去磷酸化的载体克隆技术

去磷酸基团的体系Vector17ul10xCIPbuffer2ulCIP1ul1.Incubateat37°Cfor30minutes2.Add30ulsterilewater3.Add50ulphenol/chloroformandextractyourDNAsolution.Transfertheaqueoussolutiontoanewtube.4.PrecipitatetheDNAbyadding5ul3Msodiumacetateand110ul100%ethanol.Incubateonicefor30minutes.5.Centrifugeatmaximumspeedinamicrocentrifugefor10minutesat4°C.Carefullydecantthesupernatant.6.Washthenucleicacidpelletwith80%ethanol,centrifuge2minutes,andremovetheethanol.7.Centrifugeagainfor1minute,removeresidualethanol,andairdrythepellet.8.Resuspendthepelletin8ulsterilewater.酶连体系去磷酸基的vector4ul酶切片段4ulT4DNALigase1ulBuffer1ul16℃酶连过夜