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NEB/Luna® Universal qPCR Master Mix/M3003S/1,000 rxn (10 x 1 ml)
  • NEB/Luna® Universal qPCR Master Mix/M3003S/1,000 rxn (10 x 1 ml)

NEB/Luna® Universal qPCR Master Mix/M3003S/1,000 rxn (10 x 1 ml)

价格: ¥5400.00 市场价: 9000.00
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货号: M3003S-1,000rxn(10x1ml)
品牌: NEB
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    • Description:

      Sample
       

      Rapid,sensitiveandprecisedye-basedqPCRdetectionandquantitationoftargetDNAandCDNAsequences.

      Dye-basedquantitativePCR(qPCR)usesreal-timefluorescenceofadouble-strandedDNA(dsDNA)bindingdye,mostcommonlySYBR®GreenI,tomeasureDNAamplificationduringeachcycleofaPCR.Atapointwherethefluorescencesignalisconfidentlydetectedoverthebackgroundfluorescence,aquantificationcycle,orCqvalue,canbedetermined.Cqvaluescanbeusedtoevaluaterelativetargetabundancebetweentwoormoresamples,ortocalculateabsolutetargetquantitiesinreferencetoanappropriatestandardcurve,derivedfromaseriesofknowndilutions.

      TheNEBLunaUniversalqPCRMasterMixisanoptimized2Xreactionmixforreal-timeqPCRdetectionandquantitationoftargetDNAsequencesusingtheSYBR®/FAMchannelofmostreal-timeqPCRinstruments.ItcontainsHotStartTaqDNAPolymeraseandhasbeenformulatedwithauniquepassivereferencedyethatiscompatIBLeacrossavarietyofinstrumentplatforms(includingthosethatrequireahighorlowROXreferencesignal).ItalsofeaturesdUTPforcarryoverpreventionandanon-fluorescent,visibledyetomonitorreactionsetup.ThisdyedoesnotspectrallyoverlapwithfluorescentdyesusedforqPCRandwillnotinterferewithreal-timedetection.

      Themastermixformulationissuppliedat2XconcentrationandcontainsallPCRcomponentsrequiredforamplificationandquantitationofDNAexceptprimersandDNAtemplate.GenomicDNAorcDNAofinterestcanbequantitatedwithLunaqPCR,andexistingaswellascommercialqPCRassayprimersequencescanbeused.


      Figure1:NEB’sLunaUniversalqPCRMasterMixoffersexceptionalsensitivity,reproducibilityandqPCRperformance
      qPCRtargetinghumanGAPDHwasperformedusingtheLunaUniversalqPCRMasterMixovera6-lograngeofinputtemplateconcentrations(20ng–0.2pgJurkat-derivedcDNA)with8replicatesateachconcentration.cDNAwasgeneratedfromJurkattotalRNAusingtheNEBProtoscript®IIFirstStrandcDNASynthesisKit(NEB#E6560).


      Figure2:NEB’sLunaUniversalqPCRMasterMixprovidessensitiveandaccuratedetectionandquantitationacrossawidevarietyofDNAsources
      Luna
      TheLunaUniversalqPCRMasterMixiscompatiblewithabroadrangeofgenomicDNAsources.qPCRtargetswerequantitatedwith50ng–0.5pggenomicDNAasinputusinganABI7500Fastreal-timeinstrument.GenomicDNAwaspurifiedbytypicalcolumn-basedmethods.Intheseexamples,strongperformancecanbeobservedintheamplificationofACTB(encodingβ-actin)fromMousekidneygenomicDNA,psbB(PhotosystemIICP47reactioncenterproteinPsbB)fromTobacco,andRDN18(18SribosomalRNA)fromYeast.


      Figure3:Extensiveperformanceevaluationofcommerciallyavailabledye-basedqPCRreagentsdemonstratestherobustnessandspecificityofLuna
      Luna
      qPCRreagentsfromNEBandothermanufacturersweretestedacross16–18qPCRtargetsvaryinginabundance,lengthand%GC,usingeitherJurkatgenomicDNAorJurkat-derivedcDNAasinput(10genomicDNAtargetsand8cDNAtargetsonBio-Radreal-timeinstrument,9genomicand7cDNAtargetsonABIinstrument).Foreachtestingcondition,datawascollectedby2usersandaccordingtomanufacturer’sspecifications.Resultswereevaluatedforefficiency,lowinputdetectionandlackofnon-templateamplification(whereΔCq=averageCqofnon-templatecontrol–averageCqoflowestinput).Inaddition,consistency,reproducibilityandoverallcurvequalitywereassessed(QualityScore).Bargraphindicates%oftargetsthatmetacceptableperformancecriteria(indicatedbygreenboxondotplotandQualityScore>3).ResultsforNEBandothermajormanufacturersareshown:Bio-Rad,SsoAdvanced™UniversalSYBR®GreenSupermix;Roche,FastStart™SYBRGreenMaster;Qiagen,QuantiTect®SYBRGreenPCRKit;ABI,PowerUP™SYBRGreenMasterMix;Promega®,GoTaq®qPCRMasterMix.NEB’sLunaUniversalqPCRMasterMixoutperformedallotherreagentstested.

      LearnmoreaboutourcomprehensiveqPCR/RT-qPCRtestingand“dotsinboxes”datavisualization

      Notes:

      PrimerDesignTheuseofqPCRprimerdesignsoftware(e.g.,Primer3)maximizesthelikelihoodofamplificationsuccesswhileminimizingnonspecificamplificationandprimerdimers.TargetswithbalancedGC/ATcontent(40–60%)tendtoamplifyefficiently.Wherepossible,entersufficientsequencearoundtheareaofinteresttopermitrobustprimerdesignandusesearchcriteriathatpermitcross-referenceagainstrelevantsequencedatabases(toavoidpotentialoff-targetamplification).ForcDNAtargets,itisadvisabletodesignprimersacrossknownsplicingsitesinordertopreventamplificationfromgenomicDNA.Conversely,primersdesignedtotargetintronicregionscanensureamplificationexclusivelyfromgenomicDNA.PrimerConcentrationFormosttargets,afinalconcentrationof250nM(eachprimer)willprovideoptimumperformance.Ifneeded,primerconcentrationscanbeoptimizedbetween100–500nM.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.cDNAdoesnotneedtobepurifiedbeforeadditiontotheLunareactionbutshouldbedilutedatleast1:10beforeadditiontoqPCR.ROXReferenceDyeSomereal-timeinstrumentsrecommendtheuseofapassivereferencedye(typicallyROX)toovercomewell-to-wellvariationsthatcouldbecausedbybubbles,smalldifferencesinvolume,andautofluorescencefromdustorparticulatesinthereaction.TheLunaUniversalqPCRMasterMixisformulatedwithauniversalreferencedyethatiscompatiblewithavarietyofqPCRinstrumenttypes,includingthosethatusenopassivereferencenormalizationandthosethatusealoworhighconcentrationofpassivereferencedye(ROX).Therefore,noadditionalcomponentsarerequiredtoensurecompatibilitywiththeseinstruments.CarryoverContaminationPreventionqPCRisanextremelysensitivemethod,andcontaminationinnewqPCRassayswithproductsfrompreviousamplificationreactionscancauseavarietyofissuessuchasfalsepositiveresultsandadecreaseinsensitivity.Thebestwaytopreventthis“carryover”contaminationistopracticegoodlaboratoryproceduresandavoidopeningthereactionvesselpostamplification.However,toaccommodatesituationswhereadditionalanti-contaminationmeasuresaredesired,theLunaUniversalqPCRMasterMixcontainsamixtureofdUTP/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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