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NEB/NEBNext? High-Fidelity 2X PCR Master Mix/M0541S/250 reactions
  • NEB/NEBNext? High-Fidelity 2X PCR Master Mix/M0541S/250 reactions

NEB/NEBNext? High-Fidelity 2X PCR Master Mix/M0541S/250 reactions

價格: ￥4320.00 市場價: 7200.00
品牌: NEB
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    • Description:







      ?
      TheNEBNextHigh-Fidelity2XPCRMasterMixisspecificallyoptimizedfortherobust,high-fidelityamplificationofnext-generationsequencing(NGS)libraries,regardlessofGCcontent.Thepolymerasecomponentofthemastermix,Q5??High-FidelityDNAPolymerase,isanovelThermostableDNApolymerasethatpossesses3′→5′exonucleaseactivity,andisfusedtoaprocessivity-enhancingSso7ddomain.Q5High-FidelityDNAPolymerasealsohasanultra-lowerrorrate(>100-foldlowerthanthatofTaqDNAPolymeraseand6-foldlowerthanthatofPyrococcusfuriosus(Pfu)DNAPolymerase).Thebuffercomponentofthemastermixhasbeenoptimizedforrobustamplification,evenwithGC-richamplicons.

      Figure1:ComparativeAnalysisofDifferentDNAPolymeraseswithKnownLowCoverageRegions.
      Figure 1: Comparative Analysis of Different DNA Polymerases with Known Low Coverage Regions.
      IndexedlibrarieswerepreparedfromhumanIMR90DNAandsplitintoindividualsamplesforlibraryamplification.Amplificationwasperformedusing8cyclesofPCRwithPhusionHigh-FidelityDNAPolymerase,KAPAHiFi?HotStartReadyMixorNEBNext?High-Fidelity2XPCRMasterMix.LibrariesweresequencedonanIlluminaHiSeq?2000.Tocorrectforslightdifferencesinthenumberofalignedreadsfromeachlibrary,180millionreadswererandomlyextractedfromeachdataset,representinganaveragecoverageof~6X.Thenumberofreadsoverlappingdistinctlowcoverageregionsofthehumangenome(Airdet.al.GenomeBIOLOGy,2011)areshownforeachlibrary.TheNEBNextHigh-Fidelity2XPCRMasterMixgivesthemostoptimalperformanceofthethreeenzymes/mastermixestested.
      Figure2:FidelityComparisonsofDifferentDNAPolymerases.
      Figure 2: Fidelity Comparisons of Different DNA Polymerases.
      Fidelitymeasurementsof?Taq?DNAPolymerase(inStandard?Taq?Buffer),KAPAHiFiHotStartReadyMixandNEBNextHigh-Fidelity2XPCRMasterMixweremeasuredside-by-sideinaPCR-basedmutationscreeningassayusingalacZmethodmodifiedfromKermekchievetal.,2003.Values(n≥2)areexpressedrelativetoKAPAHiFiHotStartReadyMix.TheQ5High-FidelityPolymeraseintheNEBNextHigh-Fidelity2XPCRMasterMixhasaleveloffidelity>10XhigherthanthatofthepolymeraseintheKAPAHiFiHotStartReadyMix.
      Figure3:ComparativeAnalysisofDifferentDNAPolymeraseswithGenomesofVarying%GC.
      Figure 3: Comparative Analysis of Different DNA Polymerases with Genomes of Varying % GC.
      LibrariesofH.influenza,R.palustrisorhumangenomicDNAwereamplifiedusingNEBNextHigh-Fidelity2XPCRMasterMix,PhusionHigh-FidelityPCRMasterMixwithHFBufferorKAPAHiFiHotStartPCRReadyMix,andsequencedonanIllumina?HiSeq2000.GCcoverageplotsweregenerated,with%GCcontentof100bpwindowsontheXaxis.Normalizedcoverageisindicatedbythebluecircles,windowsatGC%indicatedbytheredlines,andbasequalityatGC%indicatedbythegreenline.NEBNextHigh-Fidelity2XPCRMasterMixprovidessubstantiallyreducedbiasonallthreegenomicDNAsamples.
      Figure4:ComparativeAnalysisofDifferentDNAPolymerasesatVaryingGC%s.
      Figure 4: Comparative Analysis of Different DNA Polymerases at Varying GC %s.
      AmplifiedlibrariesofhumangenomicDNAweregeneratedusingindexprimersandNEBNextHigh-Fidelity2XPCRMasterMix,Phusion?High-FidelityPCRMasterMixwithHFBufferorKAPAHiFiHotStartReadyMix,andsequencedonanIllumina?HiSeq?2000.AnequalnumberofreadsfromeachdatasetwererandomlyselectedandthepercentageofreadswasplottedagainstGCcontent.NEBNextHigh-Fidelity2XPCRMasterMixandKAPAHiFHotStartReadyMixalignedcloselytotheexpectedreadfrequencies(shadedgrey),whilePhusiondidnot.

      ProductSource

      AnE.colistrainthatcarriestheQ5High-FidelityDNAPolymerasegene.

      ReactionVolumeDefinition

      NEBNextHigh-Fidelity2XPCRMasterMix,DNAtemplateand0.5μMto1.25μMprimers(dependingonsampleinput)inatotalreactionvolumeof50μl.

      Notes:

      Toensureoptimalperformance,themastermixshouldbethawedandresUSPendedpriortouse.StABIlitytestingusingupto20freeze/thawcycleshasshownnonegativeeffectonmastermixperformance.TheNEBNextHigh-Fidelity2XPCRMasterMixmaybeliquidat-20°C.
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