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

價格: ￥4740.00 市場價: 7900.00

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

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