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NEB/SplintR® Ligase/M0375S/6,250 units
  • NEB/SplintR® Ligase/M0375S/6,250 units

NEB/SplintR® Ligase/M0375S/6,250 units

价格: ¥4908.00 市场价: 8180.00
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货号: M0375S-6,250units
品牌: NEB
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    • Description:

      SplintRLigase,alsoknownasPBCV-1DNALigaseorChlorellavirusDNALigase,efficientlycatalyzestheligationofadjacent,single-strandedDNAsplintedbyacomplementaryRNAstrand.ThispreviouslyunreportedactivitymayenablenovelapproachesforcharacterizationofmiRNAsandmRNAs,includingSNPs.SplintRisideallysuitedformanytargetenrichmentworkflowswithapplicationsinnext-generationsequencingandmoleculardiagnostics.TherobustactivityoftheenzymeanditsaffinityforRNA-splintedDNAsubstrates(apparentKm=1nM)enablesub-nanomolardetectionofuniqueRNAspecieswithinacomplexmixture,makingSplintRligaseasuperiorchoicefordemandingRNAdetectiontechnologies.

      TheenzymeisactiveoverabroadrangeofATPconcentrations(10µM–1mM)andpH(6.5–9).OptimalactivityisobservedusingMg2+>5mM,andapHbetween7.5and8.0.Theactivityisenhancedathighertemperatures(upto37°C),andbysupplementationwith5mMMn2+.Thereactionisinhibitedbysaltconcentrationsinexcessof100mM.

      Theenzymetoleratesallbasepaircombinationsattheligationjunction,butispartiallyinhibitedbydC/GanddG/Cbasepairsatthedonor(phosphorylated)sideligationjunction,particularlywhenthe+2basewasalsoaC/Gbasepair.

      LigationofDNAsplintedbyRNALigation of DNA splinted by RNA

      (A)Outlineoftheligationassay:a5´-phosphorylated,3´-FAMlabeledDNA“donor”oligonucleotideandanunmodifiedDNA“acceptor”oligonucleotideare annealedtoacomplementaryRNAsplint.Thissubstrateisreactedwithaligasetoformamixtureofunreactedstartingmaterial(I),adenylylatedDNA(II),andligatedproduct(III).These productsaredenatured,separatedbycapillaryelectrophoresisanddetectedbyfluorescence.(B)LigationoftheRNA-splintedsubstrateinSplintRLigaseReactionBufferfor15minutesat 25°Cwith(a)noenzyme,(b)1μMT4DNALigaseand(c)100nMSplintRLigase.IndicatedpeakscorrespondtostartingpDNA(I),AppDNA(II)andligatedproduct(III)asdeterminedby co-elutionwithsyntheticallypreparedstandards.(C)ThefractionofligatedproductcatalyzedbyeitherSplintRLigaseorT4DNALigasewasanalyzedbyperformingsetsofligationswith bothligasesatconcentrationsbetween10pMand10μMfor15minutesat25°C.SplintRLigaseisclearlymuchmoreefficientatligationofRNAsplintedDNAthanT4DNALigase.

      ProductSource

      AnE.colistrainthatcarriesarecombinantgeneencodingPBCV-1DNALigase.

      ReagentsSupplied

      Thefollowingreagentsaresuppliedwiththisproduct:

      Storeat(°C)Concentration
      SplintRLigaseReactionBuffer10X

      Notes:

      SplintRLigaseisinhibitedbymonovalentcations.Westronglyrecommendensuringthesecommonreactants(NaCl,KCl)arekepttobelow50mMinthereaction.Theenzymeissuppliedinastoragebuffercontaining300mMNaCl,forstoragestABIlity.Aminimum6-folddilutionoftheenzymebyadditiontothereactionisrecommended,withtheoptimaldilutionbeing≥10-fold.Ifdilutionofenzymeforstorageisneeded,werecommendusingDiluentA(NEB#B8001).ReactionswithSplintRshouldbeperformedbetween16–37°C.Werecommendinitialtestingbeperformedat25°C.Theenzymeissuppliedasa10.5μMsolution.Wesuggestmaintainingtheenzymebelow1μMinthereactionwithasuggestedrangeof100nMto1μM.Formanyapplications,startingwitha2-foldexcessofenzymeoverligatableendsisideal.Forexample,intheexperimentdescribedintheaccompanyingfigureasubstrateconcentrationof100nMwasfoundtobeideal.Inthatworkflow,250nMenzymegavecompleteligationin15minutesonallsequencestested.Ifthereactionisnotproceedingasefficientlyasdesired,westronglyrecommendextendingtheincubationtimeratherthanincreasingtheconcentrationofenzymeinthereactionbeyond1μM.AnalternativeoptionforrecalcitrantsubstratesistousealowconcentrationofATP.AlowATPbuffercangivehigheryieldsofligationproductforsubstratesthathavelowligationefficienciesinthestandardSplintRLigaseReactionBuffer,suchassubstrateswithrunsofG:Cbasepairsattheligationjunction.Wesuggest1XT4RNALigaseReactionBuffer(NEB#B0216)supplementedwithATP(NEB#P0756)toafinalconcentrationof10μM.WerecommendanRNAsplintofatleast20complementarybases.Wehavefound10bpofdsDNA/RNAoneithersideofthejunctiontobesufficientforallsubstratestested.Thesplintdoesnothavetobecenteredontheligationjunction,however,withasfewasfourbasesononesideofthejunctiongivingcompleteligationforasplintwith20basesoftotalcomplementarity,dependingonsubstratesequence.Ifregionsofoverlap<10bparedesired,sometestingwillberequiredtodeterminetheminimumlengthofthedsregionforyourspecificsequence.InordertomaximizefidelityinligationsbySplintRDNALigase,pleaseconsiderthefollowing:IncreasingreactiontemperaturehasbeenshowntoimproveactivityandspecificityofSplintRDNALigase.Incubationat37°CgivesimprovedresultsindetectionofmicroRNA[2].AdditionofETSSB(NEB#M2401)hasalsobeenshowntoincreaseactivityandreduceoff-targetligationwhenligatingDNAprobestoRNAusingSplintRDNALigase[3].
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