荧光定量PCR引物设计软件说明书(PrimerExpress_guide)

PrimerExpressOperationGuidePrimerExpressv3.0PrimersandProbeDesignForReal-TimePCRPrimerExpressOperationGuidePrimers/ProbesDesignGuidelineTaqManProbePrimerProbePrimer離,PCR50-150bpG/C%30-80%列4GTm:68-70(Quantificationassay)65-67(AllelicDiscriminationassay)Tm:58-60Probe度:13~25bases(TaqManMGBprobe)13~30bases(TaqManprobe)Primer度:20bases(Optimal)連6A列5’列不G(FAM-dye5’列不G)CGstrandprobeb3’4列裡3G(GGG-MGB-3’orGGAG-MGB-3’)aprobe2CCdi-nucleotidesa3’列裡不2C+Ga:TaqManMGBprobeb:參數PrimerExpressOperationGuidePrimers&ProbesforQuantificationAutomaticallyDesignPrimerExpress3.0File→New→“TaqManMGBQuantificationTaqManQuantification”→OKTools→“AddDNAFile”列”Add”列”Sequence”TabCopy&Paste列*PrimerExpressSoftware.dan,.txt,.ab1,.abi,列(若列databasedownload來,列料)PrimerExpressOperationGuide”DoubleStrand“,doublestrandedDNAsequenceTools1.Exclude:不列利”ExcludeSelectedBases”列-不列“Exclude”見PrimerExpressOperationGuide2.Junction:列junction利”Junction”-juction(2bases)“Junction”見來3.”Parameters”TabPrimer/ProbeTmPrimers/ProbePrimerExpressOperationGuide”Sequence”TabTools→FindPrimers/ProbesPrimers/ProbepairsPrimerExpressCandidatePrimers&Probepairssearch50列”Primers/Probes”Tab”Location”說primers&probes列數”Sequence”Tab”Primers/Probes”TabPrimers/Probe:Probe藍ForwardPrimerReversePrimer(*不)PrimerExpressOperationGuide”Primers/Probes”TabPrimers/Probe列來2primer/probedesignguidelineMGBProbe”Parameters”Tab”C””G”列參數primer/probeset行File→SaveAsprimer/probeset利Export→OrderInfo…50Primers/ProbesExport→Primers/ProbesList…兩都.txtExcelPrimerExpressOperationGuideManuallyDesignPrimer/ProbePrimer/ProbeQuantificationDocument列(參3)1.Probe:”Sequence”TabProbe列highlight來(25bases度度列)利EditCopy(Ctrl+C)列Tools→PrimerProbeTestTooldocumenttype(“TaqManMGBQuantificationTaqManQuantification”)Parameter”Default”利Paste(Ctrl+V)列Probe1欄列Tm,%GC度:probe列不G列裡C數G若Tm(68to70)Probe1欄不列Tm,%GC度Probe(參primer/probedesignguideline)若”Trim”列留Probe列PrimerExpressOperationGuideProbe列”Sequence”TabProbe列highlight來利Edit→Annotate→”Probe”probe列來probe綠利Tools→FindPrimers/ProbesProbeForward/ReversePrimers若Primers行Forward/ReversePrimersPrimerExpressOperationGuide2.ForwardPrimer:”Sequence”TabPrimer列highlight來(25bases度切Probe列)利EditCopy(Ctrl+C)列Tools→PrimerProbeTestTool利Paste(Ctrl+V)列FwdPrimer欄列Tm,%GC度ForwardPrimerTm58-60Tm不FwdPrimer不列Tm,%GC度Primer(參primer/probedesignguideline)若”Trim”列留列FwdPrimer列”Sequence”Tab列highlight來利”ForwardPrimer”來FwdPrimer藍PrimerExpressOperationGuide3.ReversePrimer”Sequence”TabReverse列highlight來(25bases度切Probe列)利Edit→”CopyComplement”列Tools→PrimerProbeTestToolRevPrimer欄ReversePrimerTm58-60Tm不RevPrimer不列Tm,%GC度Primer(參primer/probedesignguideline)若”Trim”列留列RevPrimer列”Sequence”Tab列highlight來利”ReversePrimer”來RevPrimerPrimerExpressOperationGuideprimer/probeset利Copy&Pastetextsave來來參料document行File→SaveAsPrimerExpressOperationGuideSYBRGreenIPrimerforQuantificationAutomaticallyDesignPrimerExpress3.0File→New→“TaqManMGBQuantificationTaqManQuantification”→OKTools→“AddDNAFile”列”Add”列”Sequence”TabCopy&Paste列*PrimerExpressSoftware.dan,.txt,.ab1,.abi,列(若列databasedownload來,列料)PrimerExpressOperationGuide”DoubleStrand“,doublestrandedDNAsequenceTools1.Exclude:不列利”ExcludeSelectedBases”列-不列“Exclude”見PrimerExpressOperationGuide2.Junction:列junction利”Junction”-juction(2bases)“Junction”見來3.”Parameters”TabPrimerTmPrimersPrimerExpressOperationGuide”Sequence”TabTools→FindPrimers/ProbesPrimers/ProbepairsPrimerExpressCandidatePrimers&Probepairssearch50列”Primers/Probes”Tab若行SYBRGreenprimer不probe列參primer列”Location”說primers&probes列數”Sequence”Tab”Primers/Probes”TabPrimers/Probe:Probe藍ForwardPrimerReversePrimer(*不)PrimerExpressOperationGuide”Primers/Probes”TabPrimers/Probe列來2primer/probedesignguideline*SYBRGreenprimerprimerdimer-”Primers/Probes”Tabprimerset來PrimerSecondaryStructureHairpin,SelfDimersCrossDimers數GC例PrimerExpressOperationGuideprimersset行File→SaveAsprimersset利Export→OrderInfo…50Primers/ProbesExport→Primers/ProbesList…兩都.txtExcelPrimerExpressOperationGuideManuallyDesignPrimerPrimerQuantificationDocument列(參13)1.ForwardPrimer:”Sequence”TabPrimer列highlight來(25bases度度列)利EditCopy(Ctrl+C)列Tools→PrimerProbeTestTooldocumenttype(“TaqManMGBQuantificationTaqManQuantification”)Parameter”Default”利Paste(Ctrl+V)列FwdPrimer欄列Tm,%GC度ForwardPrimerTm58-60Tm不FwdPrimer不列Tm,%GC度Primer(參primer/probedesignguideline)若”Trim”列留列PrimerExpressOperationGuide2.ReversePrimer:”Sequence”TabReverse列highlight來(25bases度度列)利Edit→”CopyComplement”列Tools→PrimerProbeTestToolRevPrimer欄ReversePrimerTm58-60Tm不RevPrimer不列Tm,%GC度Primer(參primer/probedesignguideline)若”Trim”列留列PrimerExpressOperationGuide3.SYBRGreenprimerprimerdimer-PrimerProbeTestToolprimerset”ShowSecondaryStucture”來PrimersHairpin,SelfDimersCrossDimers數GC例primerFwdPrimer列”Sequence”Tab列highlight來利”ForwardPrimer”來FwdPrimer藍PrimerExpressOperationGuideRevPrimer列”Sequence”Tab列highlight來利”ReversePrimer”來RevPrimerprimer/probeset利Copy&Pastetextsave來來參料document行File→SaveAsPrimerExpressOperationGuidePrimers&ProbesforAllelicDiscriminationAutomaticallyDesignPrimerExpress3.0File→New→“TaqManMGBAllelicDiscriminationTaqManAllelicDiscrimination”→OK