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NEB/HiFi Taq DNA Ligase/M0647S/50 reactions
  • NEB/HiFi Taq DNA Ligase/M0647S/50 reactions

NEB/HiFi Taq DNA Ligase/M0647S/50 reactions

价格: ¥1800.00 市场价: 3000.00
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货号: M0647S-50reactions
品牌: NEB
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    • Description:

      Thermo Calc

       
      Article




      AnoptimizedblendofaThermostableDNALigaseandaproprietaryadditive,HiFiTaqDNALigaseefficientlysealsnicksinDNAwithunmatchedhighfidelity.Theformationofaphosphodiesterbondbetweenjuxtaposed5´phosphateand3´hydroxylterminioftwoadjacentoligonucleotidesthatarehybridizedtoacomplementarytargetDNAisenhancedintheimprovedreactionbufferandmismatchligationisdramaticallyreduced(1).Theimprovedformulationallowshigherresolutiondiscriminationbetweenligationdonorsandacceptors,enablingprecisedetectionofSNPsandotherallelevariants.HiFiTaqDNALigaseisactiveatelevatedtemperatures(37–75°C)(2,3).

      PleasenotethatHiFiTaqDNALigaseisintendedforuseinmoleculardiagnosticsapplicationsthatdependonhighfidelitynickligation.ItisnotasubstituteforT4DNAligaseandisnotsuitableforcloningapplicationsoradapterligation/NGSlibraryprep.

      Useouronlinetool,ThermostableLigaseReactionTemperatureCalculator,tohelpselectthecorrectincubationtemperatureforHiFiTaqDNALigase.

      Figure1:HiFiTaqDNALigasedisplaysincreasedfidelity


      (A)Schematicofmultiplexedsubstratepools.EachsubstratepoolcontainedasinglesplintwithadefinedNNattheligationjunction(e.g.,AA,AC,AG…)alongwithallfourupstreamprobesandallfourFAM-labeleddownstreamprobes.Eachprobethatencodesthebaseattheligationjunctionisofuniquelengthallowingforseparationandanalysisbycapillaryelectrophoresis.Atotalof16substratepoolswereprepared,oneforeachuniquesplint.(B)ComparisonoftheligationfidelityofAmpligase(Epicentre),TaqDNALigaseandHiFiTaqDNALigase.Fidelitymeasurementswereperformedusing1μlofligaseina50μlreactionmixtureinthesuppliedbuffersat1Xconcentration.Reactionswereincubated30minat55°C,usingmultiplexedsubstratepoolsasoutlinedin(A).Rowsrepresentasingletemplatesequence,whilecolumnsindicateaparticularligationproductresultingfromaspecificpairofprobesligatingwiththeindicatedbasesattheligationjunction.Adotindicatesdetectionofaproduct(seelegendabove).ThediagonalfromthetoplefttothebottomrightrepresentsWatson-Crickligationproducts;allotherspacesindicatemismatchligationproducts.WhileTaqDNALigaseandAmpligaseperformsimilarlyundertheseconditions,witharangeofmismatchproductsdetectable,HiFiTaqDNALigaseshowsdramaticallyfewermismatchproductswhilemaintaininghighyields(imageadaptedfromReference1).

      Figure2:HiFiTaqDNALigaseexhibitsincreaseddiscriminationatbothsidesoftheligationjunction


      Oligonucleotideprobestargetinga35bpregionoftheλintegrasegenewereincubatedwith16.7fmolofλgenomicDNA.LigationproductsformedweredetectedbyqPCRusingSYBR®Green.HiFiTaqDNALigase(NEB#M0647)displaysincreasedfidelityoverTaqDNALigase(NEB#M0208)andAmpligase,showingalargerΔCtvaluebetweenprobesfullycomplementarytothetargetsequenceandthosewithamismatchedbasepairattheligationjunction.
      Figure3:HiFiTaqDNALigaseexhibitsincreasedthermostABIlity

      HiFi Taq DNA Ligase exhibits increased thermostability

      HiFiTaqDNALigaseandAmpligase®(1µlenzymeina50µlreaction)werecycled(80°Cfor90seconds/94°Cfor10 seconds)upto100 timesintheirrespective1Xreactionbuffer.LigaseactivitywasassayedusingaFAM-labelednickeddsDNAsubstratedetectedbycapillaryelectrophoresis.
      Figure4:HiFiTaqDNALigasedisplays2-foldreductioninmismatchligationascomparedtoAmpligase

      HiFi Taq DNA Ligase displays 2-fold reduction in mismatch ligation as compared to Ampligase

      TheC:GWatson-Crickbasepairbetweentheupstreamprobe3´-terminalbaseandthecomplementarystrandmakesthisaparticularlydifficultjunctionforaligasetodiscriminateagainstmismatchligationproducts(1).

      ProductSource

      PurifiedfromanE.colistrainthatcarriestheclonedligasegenefromahyperthermophilicorganism.

      ReagentsSupplied

      Thefollowingreagentsaresuppliedwiththisproduct:

      Storeat(°C)Concentration
      HiFiTaqDNALigaseReactionBuffer-2010X

      Notes:

      ReactionConditions:ReactionswithHiFiTaqDNALigaseshouldbeperformedbetween37–75°C.Theoptimalligationincubationtemperatureforagivensetofprobesistypicallywithin5°CoftheTmoftheprobesannealingregionandmustbedeterminedempiricallyforthebestbalanceofactivityandfidelityinyourapplication.Useofthe ThermostableLigaseReactionTempCalculatorishighlyrecommendedforselectionofanincubationtemperature.ForatypicalLDR-typeassay,reactiontimeshouldbeintherangeof10–60minutes,with15minutesbeingidealformanyapplications.Forassaysrequiringdenaturation/annealing/ligationcycling,wesuggestdenaturation@95°Cfor30s–1min,followedbyannealing/ligationfor1–5minattheoptimizedligationtemperatureforyourprobeset.1XHiFiTaqDNALigaseReactionBufferrequiresNAD+asacofactor.NAD+issuppliedinthe10XHiFiTaqDNALigaseReactionBuffer.While10XHiFiTaqDNALigaseBufferisstableat-20°Cfor3years,thebuffershouldbestoredat-80°Ctofurtherextendthehalf-lifeoftheNAD+cofactor.TodiluteHiFiTaqDNALigasethatwillsubsequentlybestoredat-20°C,50%glycerolstoragebuffer(DiluentBufferA,NEB#B8001)canbeused,however,theshelflifeofHiFiTaqDNALigasedilutedinthisbufferwillbereduced.Todiluteforimmediateuse,1XHiFiTaqDNALigaseReactionBuffercanbeused.InordertoachievethehighestfidelitypossIBLewithHiFiTaqDNALigase,werecommendusingHiFiTaqDNALigaseReactionBuffer,however,HiFiTaqDNALigasecanbeusedinTaqDNALigaseReactionBufferwithreducedactivityandfidelity.Conversely,theuseofstandardTaqDNALigaseinHiFiTaqDNALigaseReactionBufferwillboostthefidelityofthatenzyme.HiFiTaqDNALigasecanbeusedinavarietyofpolymerasebufferswhensupplementedwith1mMNADwithreducedactivityandmodestlyreducedfidelity.Whenvisualizingligationreactionsbygel,someDNAsequencesmayexhibitagelshiftduetobindingofproteincomponents.Thisproteinbindingdoesnotinterferewithmostdownstreammethodsincludingamplification.Werecommendpre-treatmentwithProteinaseK(1μlinatypical50μlligationreaction)for30minat37°CbeforeloADIngonageltoremovethisshift.Themolarconcentrationoftheligaseis44nM.
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