PCR实验报告(分子生物学实验).pdf

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1、PCRLinChengyuBio042010030007;Cooperator:YuanXiaowenExperimentdate:2012-03-22SubmitDate:2012-03-231Introduction1.1BackgroundinformationPCR(polymerasechainreaction)isatechniqueforamplifyingDNAsequencesinvitro.ItwasinventedbyKaryMullisandhiscolleaguesin1985.Itiswidelyusedingeneclon

2、ing,mutation,sequencinganddetection.1.2Objectives(1)LearntheprincipleandmethodofPCR(PolymeraseChainReaction).(2)ComprehendthesignificanceofPCRtechniqueinDNAmanipulation.1.3MajorprinciplesOnetypicalPCRcycleconsistsofthreesteps:denaturation,annealingandextension.(1)DenaturationFig

3、ure1Step1:denaturationAsshowninFigure1,thetargetdouble-strandDNAcanbeseparatedintosingle-strandDNAunderhightemperature(about50℃).(2)AnnealingFigure2Step2:annealingAsshowninFigure2,whentemperatureislower,theforwardandreverseprimerswillbindtothesinglestrandDNA,whichareabout20ntlon

4、g,designedtobecomplementarytothebothendofthetargetgeneandoftenhasrestrictionenzymerecognitionsitesatthe5’ends.(3)ExtensionFigure3Step3:extensionAsshowninFigure3,whentemperaturerisetoabout72℃,whichistheoptimaltemperatureofTaq,onekindofDNApolymeraseworkinginhightemperature.Thedisc

5、overyofTaqDNApolymeraseisfundamentaltoPCR.Asaresult,newDNAstrandcomplementarytothetargetDNAwillbesynthesizeattheendof3’endsoftheprimeruntiltemperaturerisetodenaturethedoublestrandagain.2ExperimentOperation2.1Material(1)pCMV-Myc-T10(SIPAR),10ng;Figure4Plasmidprofile(pCMV–Myc)(2)F

6、orwardandreverseprimer,1µM;Figure5ForwardprimerFigure6Reverseprimer(3)DNApolymerase:rTaq,5U/µl.2.2Chemicalsandapparatus2.2.1SolutionsTable1SolutionsdNTPmixtureEach2.5mM100mMTris-HCl(pH8.3)10×PCRbuffer50mMKCl15mMMgCl20.25%Bromophenolblue3×loadingbuffer30%sucroseAgarosegel1.0g/100

7、mlagarose1×TAEbufferEB10mg/mlEthidiumbromideBindingbufferGuanidine(pH7.2)80mMKAc8.3mMTris-HCl(pH7.5)Washingbuffer40µMEDTAadd3foldsvolumeofabsoluteethanolbeforeuse2.2.2Apparatus(1)Pipettes;(2)PCRsystems;(3)Eppendorftubes;(4)Centrifuge;(5)Gelelectrophoresisapparatus;(6)UVgelimagin

8、gsystem;(7)BiodevPCRpurificationkit.2.3Procedur