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NEB/NEBNext? Ultra? II Q5? Master Mix/M0544S/250 reactions
  • NEB/NEBNext? Ultra? II Q5? Master Mix/M0544S/250 reactions

NEB/NEBNext? Ultra? II Q5? Master Mix/M0544S/250 reactions

價(jià)格: ￥4740.00 市場(chǎng)價(jià): 7900.00
貨號(hào): M0544S-250reactions
品牌: NEB
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    • Description:







      ?
      TheNEBNextUltraIIQ5?MasterMixisanewformulationofQ5DNApolymerasethathasbeenoptimizedforrobust,high-fidelityamplificationofnext-generationsequencing(NGS)libraries.Thisnewformulationfurtherimprovestheuniformityofamplificationoflibraries,includingsuperiorperformancewithGC-richregions.

      Thepolymerasecomponentofthemastermix,Q5High-FidelityDNAPolymerase,isanovelThermostableDNApolymerasethatpossesses3′→5′exonucleaseactivity,andisfusedtoaprocessivity-enhancingSso7ddomain.Q5alsohasthehighestfidelityavailable(>100-foldhigherthanthatofTaqDNAPolymeraseand~12-foldhigherthanthatofPyrococcusfuriosus(Pfu)DNAPolymerase),resultinginultra-lowerrorrates.

      TheNEBNextUltraIIQ5MasterMixisanaptamer-basedhotstartformulationthatallowsconvenientroomtemperaturereactionsetup.Theconvenient2XmastermixformatcontainsdNTPs,Mg++andaproprietarybuffer,andrequiresonlytheadditionofprimersandDNAtemplateforrobustamplification.NEBNextUltraIIQ5MasterMixisalsoincludedintheNEBNextUltraIIDNALibraryPrepKitforIllumina?.

      NEBNextUltraIIQ5MasterMixprovidesimprovedcoverageofknownlowcoverageregionsofthehumangenome.
      LibrarieswerepreparedfromHumanNA19240genomicDNA.Onelibrarywasnotamplified.Theothertwolibrarieswereamplifiedusing5cyclesofPCRwithNEBNextQ5HotStartHiFiPCRMasterMix(NEB#M0543)orwithNEBNextUltraIIQ5MasterMix(NEB#M0544).LibrariesweresequencedonanIllumina?NextSeq?500.420million75bpreadswererandomlyextractedfromeachdataset,representinganaveragecoverageof10X.ReadsweremappedtotheGRCh37referencegenomeusingBowtie2.2.4.Readsoneachregionwerecountedusingbedtoolsv2.19.1.A:Thenumberofreadsoverlappingdistinctlowcoverageregionsofthehumangenome(1)areshownforeachlibrary.B:Fromthe420million75bpreadsrandomlyextractedfromeachdataset,10Xcoveragewasexpected.The%ofdifficultregionscoveredat>10Xareshownforeachlibrary.TheNEBNextUltraIIQ5MasterMixprovidesimprovedcoverageoftheseknownlowcoverageregions,withoutdrop-outs,andshowssimilarcoveragetotheunamplifiedsample.

      UltraIIprovidesthehighestandmostuniformcoverageofdifficultsequenceregions.
      Indexedlibrarieswerepreparedfrom100ngofHumanNA19240genomicDNAusingaPCR-freeworkfloworthelibraryprepkitsshown,followingmanufacturers’recommendations.ThePCR-freelibrarywaspreparedusingNEBNextUltraII.LibrariesweresequencedontheIlluminaNextSeq?500.420millionreadswererandomlyextractedfromeachdataset,toproduceanaveragecoverageof10X.ReadsweremappedtotheGRCh37referencegenomeusingBowtie2.2.4.Readsoneachregionwerecountedusingbedtoolsv2.19.1.Thenumberofreadsoverlappingdistinctdifficult,low-coverageregionsofthehumangenome(1)areshownforeachlibrary.UltraIIprovidesthehighestandmostuniformcoverageofthesedifficultregions,andprovidesthecoverageclosesttothatobtainedwithaPCR-freeprotocol.
      NEBNextUltraIIQ5MasterMixprovidesuniformGCcoverageformicrobialgenomicDNAwithabroadrangeofGCcomposition.


      Librariesweremadeusing100ngofthegenomicDNAsshownandtheNEBNextUltraIIDNALibraryPrepKit.LibrarieswereamplifiedusingtheNEBNextUltraIIQ5MasterMix,andsequencedonanIlluminaMiSeq.GCcoverageinformationwascalculatedusingPicard’sCollectGCBiasMetrics(v1.117).Expectednormalizedcoverageof1.0isindicatedbythehorizontalgreyline,thenumberof100bpregionsateachGC%isindicatedbytheverticalgreybars,andthecoloredlinesrepresentthenormalizedcoverageforeachlibrary.NEBNextUltraIIQ5MasterMixprovidesuniformGCcoverageregardlessoftheGCcontentoftheDNA.
      StABIlity:Stabilitytestingusingupto30freeze/thawcycleshasshownnonegativeeffectonmastermixperformance.TheNEBNextUltraIIQ5MasterMixmaybestoredat–20°Cforupto18months.

      Source
      AnE.colistrainthatcarriestheQ5High-FidelityDNAPolymerasegene.

      ReactionConditions
      NEBNextUltraIIQ5MasterMix,DNAtemplateand1μMprimersinatotalreactionvolumeof50μl.
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