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NEB/Luna? Universal Probe qPCR Master Mix/M3004S/500 rxn (5 x 1 ml)
  • NEB/Luna? Universal Probe qPCR Master Mix/M3004S/500 rxn (5 x 1 ml)

NEB/Luna? Universal Probe qPCR Master Mix/M3004S/500 rxn (5 x 1 ml)

價(jià)格: ￥2676.00 市場(chǎng)價(jià): 4460.00
貨號(hào): M3004S-500rxn(5x1ml)
品牌: NEB
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    • Description:

      Sample
      ?

      Rapid,sensitiveandpreciseprobe-basedqPCRdetectionandquantitationoftargetDNAandCDNAsequences.

      Probe-basedquantitativePCR(qPCR)usesreal-timefluorescencereleasedupon5′→3′exonucleasecleavageofaquenched,target-specificprobetomeasureDNAamplificationateachcycleofaPCR.Atapointwherethefluorescencesignalissignificantlydetectableoverthebackgroundfluorescence,aquantificationcycleorCqvaluecanbedetermined.Cqvaluescanbeusedtoevaluaterelativetargetabundancebetweentwoormoresamplesortocalculateabsolutetargetquantitiesinreferencetoanappropriatestandardcurve,derivedfromaseriesofknowndilutions.

      TheNEBLunaUniversalProbeqPCRMasterMixisa2Xreactionmixoptimizedforreal-timeqPCRdetectionandquantitationoftargetDNAsequencesusinghydrolysisprobes.ItcontainsHotStartTaqDNAPolymeraseandhasbeenformulatedwithauniquepassivereferencedyethatiscompatIBLeacrossavarietyofinstrumentplatforms(includingthosethatrequireahighorlowROXreferencesignal).ItalsofeaturesdUTPforcarryoverpreventionandanon-fluorescent,visibledyetomonitorreactionsetup.ThisdyedoesnotspectrallyoverlapfluorophorescommonlyusedforqPCRandwillnotinterferewithreal-timedetection.

      Themastermixformulationissuppliedat2XconcentrationandcontainsallPCRcomponentsrequiredforamplificationandquantitationofDNAexceptprimers/probesandDNAtemplate.GenomicDNAorcDNAofinterestcanbequantitatedwithLunaqPCRandexistingaswellascommercialqPCRassayprimer/probesequencescanbeused.

      Figure1:NEB’sLunaUniversalProbeqPCRMasterMixoffersexceptionalsensitivity,reproducibilityandqPCRperformance
      qPCRtargetinghumanGAPDHwasperformedusingtheLunaUniversalProbeqPCRMasterMixovera6-lograngeofinputtemplateconcentrations(20ng–0.2pgJurkat-derivedcDNA)with8replicatesateachconcentration.cDNAwasgeneratedfromJurkattotalRNAusingtheNEBProtoscript?IIFirstStrandcDNASynthesisKit(NEB#E6560).


      Figure2:NEB’sLunaUniversalProbeqPCRMasterMixoffersrobustperformanceinmultiplexapplications
      Luna
      Singleplex(left)andmultiplex(right)qPCRstargetinghumanGAPDH,ribosomalproteinL32gandPI-3-Kinase-RelatedKinaseSMG1wereperformedusingtheLunaUniversalProbeqPCRMasterMixovera5-lograngeofinputtemplateconcentrations(20ng–2pgJurkat-derivedcDNA)with4replicatesateachconcentration.0.4μMprimerwasusedforthelower-copytarget(SMG1)and0.2μMprimerforeachhigher-copytarget(L32gandGAPDH),inbothmultiplexqPCR(toaccountforcopynumberdifferences)andsingleplexqPCR(toallowdirectcomparison).


      Figure3:Extensiveperformanceevaluationofcommerciallyavailableprobe-basedqPCRreagentsdemonstratestherobustnessandspecificityofLuna
      Luna DNA probe
      qPCRreagentsfromNEBandothermanufacturersweretestedon10qPCRtargets,varyinginabundance,lengthand%GC,usingeitherJurkatgenomicDNAorJurkat-derivedcDNAasinput(5targetseach).Foreachtestingcondition,datawascollectedby2usersandaccordingtomanufacturerspecifications.Resultswereevaluatedforefficiency,lowinputdetectionandlackofnon-templateamplification(whereΔCq=averageCqofnon-templatecontrol–averageCqoflowestinput).Inaddition,consistency,reproducibilityandoverallcurvequalitywereassessed(QualityScore).Bargraphindicates%oftargetsthatmetacceptableperformancecriteria(indicatedbygreenboxondotplotandQualityScore>3).ResultsforNEBandothermajormanufacturersareshown:Qiagen,QuantiTect?ProbePCRKit;Bio-Rad,SsoAdvanced?UniversalProbesSupermix;Roche,FastStart?TaqMan?ProbeMaster;ABI,TaqManFastAdvancedMasterMix;Promega?,GoTaq?ProbeqPCRMasterMix.NEB’sLunaUniversalProbeqPCRMasterMixoutperformedallotherreagentstested.

      LearnmoreaboutourcomprehensiveqPCR/RT-qPCRtestingand“dotsinboxes”datavisualization

      Notes:

      AssayDesignTheuseofqPCRprimerdesignsoftware(e.g.,Primer3)maximizesthelikelihoodofamplificationsuccesswhileminimizingnonspecificamplificationandprimerdimers.TargetswithbalancedGC/ATcontent(40–60%)tendtoamplifyefficiently.Wherepossible,entersufficientsequencearoundtheareaofinteresttopermitrobustprimerdesignandusesearchcriteriathatpermitcross-referenceagainstrelevantsequencedatabases(toavoidpotentialoff-targetamplification).ForcDNAtargets,itisadvisabletodesignprimersacrossknownsplicingsitesinordertopreventamplificationfromgenomicDNA.Conversely,primersdesignedtotargetintronicregionscanensureamplificationexclusivelyfromgenomicDNA.PrimerandProbeConcentrationFormosttargets,afinalconcentrationof400nMforeachprimerwillprovideoptimumperformance.Ifneeded,primerconcentrationscanbeoptimizedbetween200–900nM.Probeshouldbeincludedat200nMforbestresults.Probeconcentrationcanbeoptimizedintherangeof100–500nMifoptimizationofperformanceortargetfluorescencelevelisdesired.MultiplexingTodetectorquantitatemultipletargetsinthesameLunareaction,selectdifferentfluorophorescorrespondingtoseparatedetectionchannelsofthereal-timeinstrument.Include400nMofforwardandreverseprimerand200nMprobeforeachtargettobedetectedinthereaction,andadjustconcentrationsifnecessarybasedonperformance(primer200–900nM,probe100–500nM).WhenloADIngqPCRprotocolontothereal-timeinstrument,besuretoselecttheappropriateopticalchannels,assomeinstrumentshaveasinglechannelrecordingmodethatwouldpreventmultiplexdatacollectionandanalysis.ForROX-dependentinstruments,avoidROX-labeledprobes.Thefunctionalityoftheprimerandprobesetsshouldbetestedindividuallybeforeattemptingamultiplexreaction.Whendeterminingwhichfluorophorestoincludeinamultiplexreaction,besuretochoosecompatiblereporterdyesandquenchersthathavewellseparatedfluorescencespectraorexhibitminimaloverlap.AmpliconLengthToensuresuccessfulandconsistentqPCRresults,itisimportanttomaximizePCRefficiency.AnimportantaspectofthisisthedesignofshortPCRamplicons(typically70–200bp).Someoptimizationmayberequired(includingtheuseoflongerextensiontimes),fortargetsthatexceedthatrange.TemplatePreparationandConcentrationLunaqPCRiscompatiblewithDNAsamplespreparedthroughtypicalnucleicacidpurificationmethods.PreparedDNAshouldbestoredinanEDTA-containingbuffer(e.g.,1XTE)forlong-termstability,anddilutionsshouldbefreshlypreparedforaqPCRexperimentbydilutionintoeitherTEorwater.Generally,ausefulconcentrationofstandardandunknownmaterialwillbeintherangeof106copiesto1copy.ForgDNAsamplesfromlargegenomes(e.g.,human,mouse)arangeof50ng–1pgofgDNAistypical.Forsmallgenomes,adjustasnecessaryusing106?–1copyinputasanapproximaterange.Notethatforsinglecopydilutions,somesampleswillcontainmultiplecopiesandsomewillhavenone,asdefinedbythePoissondistribution.ForcDNA,usetheproductofareactioncontaining1μg–0.1pgstartingRNA.cDNAdoesnotneedtobepurifiedbeforeadditiontotheLunareactionbutshouldbedilutedatleast1:10intotheqPCR.ROXReferenceDyeSomereal-timeinstrumentsrecommendtheuseofapassivereferencedye(typicallyROX)toovercomewell-to-wellvariationsthatcouldbecausedbybubbles,smalldifferencesinvolume,andautofluorescencefromdustorparticulatesinthereaction.TheLunaUniversalProbeqPCRMasterMixisformulatedwithauniversalreferencedyethatiscompatiblewithavarietyofqPCRinstrumenttypes,includingthosethatusenopassivereferencenormalizationandthosethatusealoworhighconcentrationofpassivereferencedye(ROX).Therefore,noadditionalcomponentsarerequiredtoensurecompatibilitywiththeseinstruments.CarryoverContaminationPreventionqPCRisanextremelysensitivemethod,andcontaminationinnewqPCRassayswithproductsfrompreviousamplificationreactionscancauseavarietyofissuessuchasfalsepositiveresultsandadecreaseinsensitivity.Thebestwaytopreventthis“carryover”contaminationistopracticegoodlaboratoryproceduresandavoidopeningthereactionvesselpostamplification.However,toaccommodatesituationswhereadditionalanti-contaminationmeasuresaredesired,theLunaUniversalProbeqPCRMasterMixcontainsamixtureofdUTP/dTTPthatresultsintheincorporationofdUintotheDNAproductduringamplification.PretreatmentofqPCRexperimentswithuracilDNAglycosylase(UDG)willeliminatepreviously-amplifieduracil-containingproductsbyexcisingtheuracilbasetoproduceanon-amplifiableDNAproduct.TheuseofaThermolabileUDGisimportant,ascompleteinactivationoftheUDGisrequiredtopreventdestructionofnewlysynthesizedqPCRproducts.Toenablecarryoverprevention,0.025units/μlAntarcticThermolabileUDG(NEB#M0372)shouldbeaddedtothereactionmix.Tomaximizeeliminationofcontaminatingproducts,setuptheqPCRexperimentsatroomtemperatureorincludea10minuteincubationstepat25°Cbeforetheinitialdenaturationstep.ReactionSetupandCyclingConditionsDuetothehotstartnatureofthepolymerase,itisnotnecessarytopreheatthethermocyclerpriortouseorsetupreactionsonice.For96-wellplates,werecommendafinalreactionvolumeof20μl.For384-wellplates,afinalreactionvolumeof10μlisrecommended.Whenprogramminginstrumentcyclingconditions,ensureaplatereadisincludedattheendoftheextensionstep,andadenaturation(melt)curveaftercyclingiscompletetoanalyzeproductspecificity.Amplificationfor40cyclesissufficientformostapplications,butforverylowinputsamples45cyclesmaybeused.
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