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NEB/Q5? Site-Directed Mutagenesis Kit/E0554S/10 reactions
  • NEB/Q5? Site-Directed Mutagenesis Kit/E0554S/10 reactions

NEB/Q5? Site-Directed Mutagenesis Kit/E0554S/10 reactions

價格: ￥2256.00 市場價: 3760.00
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    • Description:

      TheQ5?Site-DirectedMutagenesisKitenablesrapid,site-specificmutagenesisofdouble-strandedplasmidDNAinlessthan2hours(Figure1).ThekitutilizestherobustQ5HotStartHigh-FidelityDNAPolymerasealongwithcustommutagenicprimerstocreateinsertions,deletionsandsubstitutionsinawidevarietyofplasmids.AfterPCR,theamplifiedmaterialisaddeddirectlytoauniqueKinase-Ligase-DpnI(KLD)enzymemixforrapid(5minutes),roomtemperaturecircularizationandtemplateremoval(Figure2).Transformationintohigh-efficiencyNEB5-alphaCompetentE.coli,providedwiththekit,ensuresrobustresultswithplasmidsuptoatleast20kbinlength.

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

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

      Thiskitisdesignedforrapidandefficientincorporationofinsertions,deletionsandsubstitutionsintodoublestranded?plasmidDNA.ThefirststepisanexponentialamplificationusingstandardprimersandamastermixfomulationofQ5HotStartHigh-FidelityDNAPolymerase.Thesecondstepinvolvesincubationwithaunique?enzymemixcontainingakinase,aligaseandDpnI.Together,theseenzymesallowforrapidcircularizationofthe?PCRproductandremovalofthetemplateDNA.Thelaststepisahigh-efficiencytransformationintochemicallycompetent?cells(provided).
      Figure3:PrimerDesignfortheQ5Site-DirectedMutagenesisKitFigure 3: Primer Design for the Q5 Site-Directed Mutagenesis Kit

      Substitutions,deletionsandinsertionsareincorporatedintoplasmidDNAthroughtheuseofspecificallydesigned?forward(black)andreverse(red)primers.Unlikekitsthatrelyonlinearamplification,primersdesigned?fortheQ5Site-DirectedMutagenesisKitshouldnotoverlaptoensurethatthebenefitsof
      exponentialamplificationarerealized.      A)Substitutionsarecreatedbyincorporatingthedesirednucleotide?change(s)(denotedby*)inthecenteroftheforwardprimer,includingatleast10complementarynucleotideson?the3′sideofthemutation(s).Thereverseprimerisdesignedsothatthe5′endsofthetwoprimersannealback-to-?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
      NEB®5-alphaCompetentE.coli(HighEfficiency)-80
      pUC19Vector-200.05ng/μl
      SOCOutgrowthMedium4

      Notes:

      StorageNote:TheQ5Site-DirectedMutagenesisKitisstableat–80°Cforoneyear.Forconvenience,theQ5HotStartHigh-Fidelity2XMasterMix,KLDEnzymeMix,KLDReactionBuffer,ControlPrimersandTemplateDNAarepackagedtogetherinaseparateboxthatcanberemovedandstoredat–20°Cfortwoyearswithnolossofactivity.TheSOCcanberemovedandstoredatroomtemperature.ItisimportanttostoretheNEB5-alphaCompetentE.coliat–80°C,andavoidrepeatedfreeze-thawcycles.
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