Youwillneed: 10xTaqbuffer(Promega)TaqPolymerase(Promega)Phenol/chloroformmix100mMdTTPTEbufferAbsoluteethanol70%ethanol 1)Digestplasmidtobeusedwithablunt-endrestrictionenzyme(IuseEcoRVandpBluescriptSK-). 2)Cleandigestionreactionbyphenol/chloroformextractionandethanolprecipitation. 3)RedissolveDNAin1xTaqPCRbuffer(IuseTaqandbufferfromPromegabutIthinkanynon-proofreADIngTaqwillwork)togiveapprox.1ugDNA/20ul. 4)AdddTTPtoafinalconc.of2mM(typicallyfroma100mMstocksolution). 5)AddTaqpolymerasetogive1unit/ugDNA/20ul. 6)Incubatemixtureat72°Cfor2hours. 7)Extractmixtureoncewithphenol/chloroformandoncewithchloroform. 8)EthanolprecipitateDNA,washwith70%ethanol,dryandredissolveinTEatanapprox.conc.of25ng/ul. 9)QuantitateDNA(Iuseanapprox.quantitationbygelfluorescenceagainststandards). 10)Forligations,useapprox.25-50ngT-vectorandsufficientPCRproducttogivea3:1,insert:vectormolarratio(IuseT4LigasefromGibco-BRLtogetherwiththeirPEG-basedbuffer). 11)LigateO/Nat15°C. 12)Transform1/5ligationmixintocompetentE.coli(IuseDH5acellsatapprox.5x107cfu/ugpUC19).Constructionofhomemade"T-vectors"
ThismethodisafterMarchuk,D.,etal.,1991,Nucl.AcidsRes.19(5),pp1154.
