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ID:52999871
大小:1.52 MB
页数:8页
时间:2020-04-15
《反转录PCR原理步骤小结.ppt》由会员上传分享,免费在线阅读,更多相关内容在PPT专区-天天文库。
1、RT-PCRforHumanTissuePrincipleofReverseTranscription-PolymeraseChainReaction(RT-PCR)RT-PCRisusedtocloneexpressedgenesbyreversetranscribingtheRNAofinterestintoitscomplementaryDNA(cDNA)throughtheuseofreversetranscriptase.Subsequently,thenewlysynthesizedcDNAisamplifiedusingtraditi
2、onalPCR.GeneralProtocolGeneralPrincipleRNAExtract&QuantificationHumanTissueTissueRNAPrepmateTMusedforRNAextrationQubitFluorometer&Quant-iTRNAKitusedforquantificationProtocolofReverseTranscription-PolymeraseChainReaction(RT-PCR)Intheone-stepprotocol,thecomponentsofRTandPCRaremi
3、xedinasingletubeatthesametime.Theone-stepprotocolgenerallyworkswellforamplifyingtargetsthatarereasonablyabundant.Alternatively,RT-PCRcanbedoneintwosteps,firstwiththereversetranscriptionandthenthePCR.Thetwo-stepprotocolisusuallymoresensitivethantheone-stepmethod;yieldsofraretar
4、getsmaybeimprovedbyusingthetwo-stepprocedure.Werecommendthetwo-stepprotocolforthisclass.MostmRNAcontainsalongstretchofadenosineresidues(AAAn)atthe3′endofthemessage;therefore,investigatorsannealaprimerconsistingof15deoxythymidineresidues(dT15)tothe3′endofthemessage.Reversetrans
5、criptasethentranscribesacomplementaryDNAstrand,startingatthedT15primer.ThecDNAcanbeisolatedbyraisingthepHofthesolution,therebydenaturingthedouble-strandedhybridandcleavingtheRNA.PrincipleofcDNAPreparationReverseTranscriptionProceduresofcDNAPreparationAssemblethereactionappropr
6、iateina0.25mlPCRtubeincluding:RNAtemplateNonspecificprimerReversetranscriptaseDNApolymeraseBufferreagentMixgently,spinbriefly.Incubateinthethermacyclerat:44°Cfor1h92°Cfor10mintoinactivatethereversetranscriptase.Storereactionat-20℃orproceedtoPCRNotetowearglovesandmaskatalltimes
7、toavoidRNasecontamination.PolymeraseChainReaction(PCR)PrincipleofPCRCopyright@2012byHIBELGAUFTS.Allrightsreserved.ThefirststepofthePCRreactionseparatesthedoublestrandsintosinglestrands(strands1and2)byheatdenaturation.Thesecondstepisaprocesscalledprimerannealing,i.e.,theformati
8、onofpartiallydouble-strandedregionsbyinteractionwiththeprimer
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