ABI7500操作手册

GettingStartedGuideRelativeQuantificationAppliedBiosystems7300/7500/7500FastReal-TimePCRSystemIntroductionandExampleRQExperimentPerformingReverseTranscriptionDesigninganRQExperimentGeneratingDatafromRQPlates-StandardGeneratingDatainanRQStudyPrimerExtendedonmRNA5′3′ReversePrimer5′Synthesisof1stcDNAstrandcDNAOligod(T)orrandomhexamer3′5′cDNASTANDARDFASTGeneratingDatafromRQPlates-FastSystemiiRelativeQuantificationGettingStartedGuidefortheAppliedBiosystems7300/7500/7500FastReal-TimePCRSystem©Copyright2004,AppliedBiosystems.Allrightsreserved.ForResearchUseOnly.Notforuseindiagnosticprocedures.AuthorizedThermalCyclerThisinstrumentisanAuthorizedThermalCycler.Itspurchasepriceincludestheup-frontfeecomponentofalicenseunderUnitedStatesPatentNos.4,683,195,4,683,202and4,965,188,ownedbyRocheMolecularSystems,Inc.,andundercorrespondingclaimsinpatentsoutsidetheUnitedStates,ownedbyF.Hoffmann-LaRocheLtd,coveringthePolymeraseChainReaction(PCR)processtopracticethePCRprocessforinternalresearchanddevelopmentusingthisinstrument.TherunningroyaltycomponentofthatlicensemaybepurchasedfromAppliedBiosystemsorobtainedbypurchasingAuthorizedReagents.ThisinstrumentisalsoanAuthorizedThermalCyclerforusewithapplicationslicensesavailablefromAppliedBiosystems.ItsusewithAuthorizedReagentsalsoprovidesalimitedPCRlicenseinaccordancewiththelabelrightsaccompanyingsuchreagents.PurchaseofthisproductdoesnotitselfconveytothepurchaseracompletelicenseorrighttoperformthePCRprocess.FurtherinformationonpurchasinglicensestopracticethePCRprocessmaybeobtainedbycontactingtheDirectorofLicensingatAppliedBiosystems,850LincolnCentreDrive,FosterCity,California94404.DISCLAIMEROFLICENSE:Norightsforanyapplication,includinganyinvitrodiagnosticapplication,areconveyedexpressly,byimplicationorbyestoppelunderanypatentorpatentapplicationsclaiminghomogeneousorreal-timedetectionmethods,includingpatentscoveringsuchmethodsusedinconjunctionwiththePCRprocessorotheramplificationprocesses.The5'nucleasedetectionassayandcertainotherhomogeneousorreal-timeamplificationanddetectionmethodsarecoveredbyUnitedStatesPatentNos.5,210,015,5,487,972,5,804,375and5,994,056,ownedbyRocheMolecularSystems,Inc.;bycorrespondingpatentsandpatentapplicationsoutsidetheUnitedStates,ownedbyF.Hoffmann-LaRocheLtd;andbyUnitedStatesPatentNos.5,538,848and6,030,787,andcorrespondingpatentsandpatentapplicationsoutsidetheUnitedStates,ownedbyAppleraCorporation.Purchaseofthisinstrumentconveysnolicenseorrightundertheforegoingpatents.UseoftheseandotherpatentedprocessesinconjunctionwiththePCRprocessrequiresalicense.Forinformationonobtaininglicenses,contacttheDirectorofLicensingatAppliedBiosystems,850LincolnCentreDrive,FosterCity,California94404,orTheLicensingDepartment,RocheMolecularSystems,Inc.,1145AtlanticAvenue,Alameda,California,94501,USA.TrademarksAppliedBiosystems,MicroAmp,PrimerExpress,andVICareregisteredtrademarksandAB(Design),ABIPRISM,Applera,CeleraGenomics,FAM,ROX,iScience,iScience(Design),andMultiScribearetrademarksofAppleraCorporationoritssubsidiariesintheUSand/orcertainothercountries.AmpErase,AmpliTaqGold,andTaqManareregisteredtrademarksofRocheMolecularSystems,Inc.SYBRGreenisaregisteredtrademarkofMolecularProbes,Inc.MicrosoftandWindowsareregisteredtrademarksofMicrosoftCorporation.Allothertrademarksarethesolepropertyoftheirrespectiveowners.PartNumber4347824Rev.C12/2004RelativeQuantificationGettingStartedGuidefortheAppliedBiosystems7300/7500/7500FastReal-TimePCRSystemiii3′cDNAcDNAPrimerExtendedonmRNA5′3′ReversePrimer5′Synthesisof1stcDNAstrandcDNAOligod(T)orrandomhexamer3′5′cDNAChapterChapterChapterChapterChapter12356STANDARDFASTIntroductionandExampleRQExperimentDesigninganRQExperimentPerformingReverseTranscriptionPrimerExtendedonmRNA5′3′ReversePrimer5′Synthesisof1stcDNAstrandcDNAOligod(T)orrandomhexamer3′5′cDNAGeneratingDatafromRQplates-FastPerforminganRQStudyAboutthe7300/7500/7500FastsystemAboutrelativequantificationAboutRQexperimentsExampleRQExperimentCreateanewRQStudydocumentConfigureanalysissettingsAnalyzeandviewresultsAdjustthebaselineandthreshholdSelectthePCRmethodSpecifythecomponentsofanRQexperimentSelectthechemistrySelectone-steportwo-stepRT-PCRChoosetheprobesandprimersIsolatetotalRNAAdjustRNAconcentrationConverttotalRNAtocDNAProgramthethermalcyclingconditionsCreateanewRQPlatedocumentPreparethereactionplateSavetheRQPlatedocumentPreparethePCRmastermixStarttherunStarttherunIfnecessary,omitsamplesExportAQPlatedocument,ifdesiredChapter4GeneratingDatafromRQplates-StandardSTANDARDFASTRQExperimentWorkflowivRelativeQuantificationGettingStartedGuidefortheAppliedBiosystems7300/7500/7500FastReal-TimePCRSystemContentsRelativeQuantificationGettingStartedGuidefortheAppliedBiosystems7300/7500/7500FastReal-TimePCRSystemvRQExperimentWorkflowiiiPrefaceviiHowtoUseThisGuide................................................viiHowtoObtainMoreInformation........................................viiiHowtoObtainServicesandSupport.....................................viiiSendUsYourComments...............................................ixCha