Gateway construct protocol.doc

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1、Gatewayconstructprotocol1.designtheprimersforyournewconstructsPrimersets:FirstattB1-for:5’-AAAAAGCAGGCTNN-Templatespecificsequences-3’attB2-rev:5’-AGAAAGCTGGGTN-Templatespecificsequences-3’SecondattB1-ad:5’-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3’attB2-ad:5’-GGGGACCACTTTGTACAAGAAAGCTGGGT-3’recombine

2、.Site2.RunthefirstPCRwithattB1-forandattB2-rev,followingtheprogramme,toamplifythetargetsequence.PCRrxmixTemplate0.2ulPrimer(F/R)0.4uldNTP(10mM)0.2ulBuffer(pfu)1.0ulEnzyme(pfu)0.1ulDD.Wtofinal10ulPCRcondition:95℃2min95℃20sec55℃40sec72℃2min/kbgotostep2,by15cycles72℃5-10min4℃forever-àcheckthePC

3、Rproduct(2-4ul/10ul)onagarosegel.3.RunthesecondPCRwithattB1-adandattB2-ad,followingtheprogramme,toamplifytherecombinationsiteandtargetsequence.PCRrxmixTemplate0.4ulPrimer(F/R)0.8uldNTP(10mM)0.8ulBuffer(pfu)2.0ulEnzyme(pfu)0.2ulDD.Wtofinal20ulPCRcondition:95℃2min95℃20sec45℃40sec72℃2min/kbgoto

4、step2by5cycles95℃20sec55℃40sec72℃2min/kbgotostep6by15cycles72℃5-10min4℃forever-àcheckthePCRproduct(2-4ul)onagarosegel.4.PurifythePCRproductwithgel-extractionorPCRelutionkit1)ifyougetonesingleband,youmayuse“PCRpurificationkit”.2)ifyougetmorethanoneband,youshoulddo“gel-extraction”.-àchecktheel

5、utedPCRproductonagarosegel5.MeasurethegeneconcentrationwithInvitrogenFluorometer6.DoBPreaction:???lattB-PCRproduct(finalamount10-30ng)xuldonorvector20-50ng)yulTEbuffer(pH8.0)to1.6ullThaw(解冻)BPclonaseIImixonice(2~3min),thenvortexingtheenzymemixbrieflytwice,and0.4ultheenzymemix.lMixwellbyvorte

6、xingbrieflytwice,shortspin,andincubate1hr~O/Nat25℃lAdd0.2uloftheProteinaseKsol’ntothesample,vortexbriefly,andthenincubateat37℃10min7.TransformationTransform1uloftheBPreactioninto50ulofDH5acellsbyelectroporation（电穿孔）.Incubatetransformantcellsat37℃1hr8.Spread200-400ulofthetransformantcellsonse

7、lectiveLBplate,transfertheplateto37℃chamberroom,O/N.9.Inoculation(mini-culture)Pick1~severalcolonies,theninoculatethemeachintoselectiveLBsol’n.Incubateat37℃O/N.10.ChecktheclonepatternslDoplasmidpreparationusingkitlDigesttheplasmidswithBsrGIlIfposit