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NEB/HiFi Taq DNA Ligase/M0647S/50 reactions

價格: ￥1800.00 市場價: 3000.00

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貨號: M0647S-50reactions

品牌: NEB

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- Description:
  
  ?
  
  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
  
  HiFiTaqDNALigaseandAmpligase?(1μlenzymeina50μlreaction)werecycled(80°Cfor90seconds/94°Cfor10?seconds)upto100?timesintheirrespective1Xreactionbuffer.LigaseactivitywasassayedusingaFAM-labelednickeddsDNAsubstratedetectedbycapillaryelectrophoresis.
  Figure4:HiFiTaqDNALigasedisplays2-foldreductioninmismatchligationascomparedtoAmpligase
  
  TheC:GWatson-Crickbasepairbetweentheupstreamprobe3′-terminalbaseandthecomplementarystrandmakesthisaparticularlydifficultjunctionforaligasetodiscriminateagainstmismatchligationproducts(1).
  ProductSource
  PurifiedfromanE.colistrainthatcarriestheclonedligasegenefromahyperthermophilicorganism.
  ReagentsSupplied
  Thefollowingreagentsaresuppliedwiththisproduct:
  Storeat(°C) Concentration
  HiFiTaqDNALigaseReactionBuffer -20 10X
  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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NEB/HiFi Taq DNA Ligase/M0647S/50 reactions

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	Storeat(°C)	Concentration
HiFiTaqDNALigaseReactionBuffer	-20	10X