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NEB/Q5??Site-Directed Mutagenesis Kit (Without Competent Cells)/E0552S/10 reactions
  • NEB/Q5??Site-Directed Mutagenesis Kit (Without Competent Cells)/E0552S/10 reactions

NEB/Q5??Site-Directed Mutagenesis Kit (Without Competent Cells)/E0552S/10 reactions

價格: ￥1572.00 市場價: 2620.00
品牌: NEB
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    • Description:

      TheQ5Site-DirectedMutagenesisKit(WithoutCompetentCells)enablesrapid,site-specificmutagenesisofdouble-strandedplasmidDNAinlessthan2hours(Figure1).ThekitutilizestherobustQ5HotStartHigh-FidelityDNAPolymerasealongwithcustommutagenicprimerstocreateinsertions,deletionsandsubstitutionsinawidevarietyofplasmids.AfterPCR,theamplifiedmaterialisaddeddirectlytoauniqueKinase-Ligase-DpnI(KLD)enzymemixforrapid(5minutes),roomtemperaturecircularizationandtemplateremoval(Figure2).Transformationintohigh-efficiencychemically-competentE.coli,notsupplied,ensuresrobustresultswithplasmidsuptoatleast20kbinlength.

      Figure1:Site-specificmutagenesisproceedsinlessthan2hours.Figure 1: Site-specific mutagenesis proceeds in less than 2 hours.

      Theuseofamastermix,auniquemulti-enzymeKLDenzymemix,andafastpolymeraseensuresthat,formost?plasmids,themutagenesisreactioniscompleteinlessthantwohours.
      ???
      Figure2:Q5Site-DirectedMutagenesisOverview.Figure 2: Q5 Site-Directed Mutagenesis Overview.

      Thiskitisdesignedforrapidandefficientincorporationofinsertions,deletionsandsubstitutionsintodoublestranded?plasmidDNA.Thefirststepisanexponentialamplificationusingstandardprimersandamastermix?fomulationofQ5HotStartHigh-FidelityDNAPolymerase.Thesecondstepinvolvesincubationwithaunique?enzymemixcontainingakinase,aligaseandDpnI.Together,theseenzymesallowforrapidcircularizationofthe?PCRproductandremovalofthetemplateDNA.Thelaststepisahigh-efficiencytransformationintochemicallycompetent?cells(notprovided).
      Figure3:PrimerDesignforQ5Site-DirectedMutagenesisFigure 3: Primer Design for Q5 Site-Directed Mutagenesis

      Substitutions,deletionsandinsertionsareincorporatedintoplasmidDNAthroughtheuseofspecificallydesigned?forward(black)andreverse(red)primers.Unlikekitsthatrelyonlinearamplification,primersdesigned?fortheQ5Site-DirectedMutagenesisKitshouldnotoverlaptoensurethatthebenefitsof?exponentialamplificationarerealized.A)Substitutionsarecreatedbyincorporatingthedesirednucleotide?change(s)(denotedby*)inthecenteroftheforwardprimer,includingatleast10complementarynucleotideson?the3′sideofthemutation(s).Thereverseprimerisdesignedsothatthe5′endsofthetwoprimersannealbackto-back.B)Deletionsareengineeredbydesigningstandard,non-mutagenicforwardandreverseprimersthatflank?theregiontobedeleted.C)Insertionslessthanorequalto6nucleotidesareincorporatedintothe5′endofthe?forwardprimerwhilethereverseprimerannealsback-to-backwiththe5′endofthecomplementaryregionofthe?forwardprimer.D)Largerinsertionscanbecreatedbyincorporatinghalfofthedesiredinsertionintothe5′ends?ofbothprimers.Themaximumsizeoftheinsertionislargelydictatedbyoligonucleotidesynthesislimitations.
      Figure4:NEB’sQ5SDMKitdelivershighertransformationefficiencythanAgilent’sQuikChange?SDMKit
      Q5 Graph
      Resultsfromasubstitutionreaction(4nt)usingtheback-to-backControlSDMPrimerMixandControlSDMPlasmid(6.7kb)areshown,alongwithresultsfroma12ntdeletionexperiment(5.8kbplasmid)andan18ntinsertionexperiment(7.0kbplasmid).Inallthreecases,over90%oftheresultantcolonieshadincorporatedthedesiredmutation(s).Resultsarenormalizedtototaltransformantsifcellswerenotdilutedpriortoplating.Forcomparison,thesamesubstitutionreaction(4nt)wasperformedwiththeQuikChangeLightningSite-DirectedMutagenesisKit(Agilent)followingAgilent’sprotocolandusingAgilent’sprimerdesigntooltodesignoverlappingprimers.

      *NotethattheQuikChangekitdoesnotaccommodatedeletionsandinsertionsofthissize,sonocomparisoncouldbemadefortheseexperiments.

      KitComponents

      Thefollowingreagentsaresuppliedwiththisproduct:

      Storeat(°C)Concentration
      Q5®HotStartHigh-Fidelity2XMasterMix-202X
      KLDEnzymeMix-2010X
      KLDReactionBuffer-202X
      ControlSDMPrimerMix-2010μM
      ControlSDMPlasmid-205μg/ml

      Notes:

      StorageNote:TheQ5Site-DirectedMutagenesisKit(WithoutCompetentCells)isstableat–20°Cfortwoyears.Forflexibility,themutagenesisreagentsandcontrolreactionsaresuppliedwithoutcompetentcellssothatanychemically-competentE.colicellssuitableforcloningmaybeused.
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