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NEB/NEBuilder? HiFi DNA Assembly Master Mix/E2621S/250 reactions
  • NEB/NEBuilder? HiFi DNA Assembly Master Mix/E2621S/250 reactions

NEB/NEBuilder? HiFi DNA Assembly Master Mix/E2621S/250 reactions

價(jià)格: ￥30240.00 市場(chǎng)價(jià): 50400.00
貨號(hào): E2621S-250reactions
品牌: NEB
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    • Description:






      NEBuilderHiFiDNAAssemblyMasterMixwasdevelopedtoimprovetheefficiencyandaccuracyofDNAassembly.ThismethodallowsforseamlessassemblyofmultipleDNAfragments,regardlessoffragmentlengthorendcompatibility.Thismethodhasbeenusedtoassembleeithersingle-strandedoligonucleotidesordifferentsizesofDNAfragmentswithvariedoverlaps(15–80bp).IthasutilityforthesyntheticBIOLOGycommunity,aswellasthoseinterestedinone-stepcloningofmultiplefragmentsduetoitseaseofuse,flexibilityandsimplemaster-mixformat.Thereactionincludesdifferentenzymesthatworktogetherinthesamebuffer(seeFigure1):
      • Theexonucleasecreatessingle-stranded3′overhangsthatfacilitatetheannealingoffragmentsthatsharecomplementarityatoneend(theoverlapregion)
      • Thepolymerasefillsingapswithineachannealedfragment
      • TheDNAligasesealsnicksintheassembledDNA?
      Theendresultisadouble-strandedfullysealedDNAmoleculethatcanserveastemplateforPCR,RCAoravarietyofothermolecularbiologyapplications,includingdirecttransformationofEcoli.

      NEBuilderHiFiDNAAssemblykitsareavailableinvariousformats:withNEB5-alphacompetentcells(CloningKit,NEB#E5520),asabundlewithNEB10-betacompetentcells(BundleforLargeFragments,NEB#E2623)andwithoutcompetentcells(MasterMix,NEB#E2621).NEB5-alphacompetentcellscellsareexcellentforroutineassembliesof15kborless.NEBrecommendsNEB10-betaCompetentE.coli(HighEfficiency,NEB#C3019)orNEB10-betaElectrocompetentE.coli(NEB#C3020)forassemblieslargerthan15kb.Iftheassembledgenescontainrepetitivesequences,NEBStableCompetentE.coli(NEB#C3040)shouldbeused.?

      NEBuilderhasbeenusedinvariousapplications,including:
      • Site-directedmutagenesis
      • ConstructionofansgRNA-Cas9expressionvector|Animation
      • Assemblyoflinearyeastexpressioncassettes
      TohelpselectthebestDNAassemblymethodforyourneeds,pleaseuseourSyntheticBiology/DNAAssemblySelectionChart.

      ForhelpdesigningyourprimersforusewithNEBuilder,pleaseviewourprimerdesignvideo.

      Figure1:OverviewoftheNEBuilderHiFiDNAAssemblyMethodFigure 1: Overview of the NEBuilder HiFi DNA Assembly Method

      Figure2:NEBuilderHiFiDNAAssemblyoffersimprovedefficiencyandaccuracyover
      NEBGibsonAssembly
      Reactionsweresetupina2-and6-fragmentassemblyreactionaccordingtorecommendedreactionconditions.NEBuilderHiFiDNAAssemblyresultsinlargernumbersofcoloniesoverNEBGibsonAssembly,forboth2-and6-fragmentassemblies.

      ViewadditionalperformancedatacomparedtoNEBGibsonAssembly
      Figure3:NEBuilderHiFidelivershighercolonyyieldthanIn-FusionHD
      Two-fragmentreactionsweresetupusingthepositivecontrolfromtheIn-FusionHDCloningKit(ClontechTakaraBioUSA,Inc),accordingtorecommendedprotocols.2μlofassemblyreactionwastransformedintosuppliedcompetentcells.1/50ofoutgrowthwasspreadonanApRplate.

      ViewadditionalperformancedatacomparedtoIn-FusionHD

      ?
      table iconComparisonofDNAAssemblyReactionTypes



      ?

      KitComponents

      Thefollowingreagentsaresuppliedwiththisproduct:

      Storeat(°C)Concentration
      NEBuilder?High-FidelityMasterMix-20
      NEBuilder?PositiveControl-202X

      Notes:

      ToensurethesuccessfulassemblyandsubsequenttransformationofassembledDNAs,NEBrecommendsthefollowing:DNA:PCRproductpurificationisnotnecessaryifthetotalvolumeofallPCRproductsis20%orlessoftheassemblyreactionvolume.HighervolumesofPCRproductsmayreducetheefficiencyofhigh-fidelityDNAassemblyandtransformationduetotheelevatedcarryoveramountsofPCRreactionbufferandunusedprimerspresentinthePCRproduct.ColumnpurificationofPCRproductsmayincreasetheefficiencyofbothhigh-fidelityDNAassemblyandtransformationby2–10foldandishighlyrecommendedwhenperformingassembliesofthreeormorePCRfragmentsorassemblinglongerthan5kbfragments.PurifiedDNAforassemblycanbedissolvedinddH2O(Milli-Q?waterorequivalentispreferable),TEorotherdilutionbuffers.Insert:Whendirectlyassemblingfragmentsintoacloningvector,theconcentrationofassemblyfragmentsshouldbeatleast2timeshigherthantheconcentrationofvector.Forassemblyof4ormorefragmentsintoavector,werecommendusinganequimolarratiooffragments.Transformation:NEB5-alphaCompetentE.coli(HighEfficiency,NEB#C2987)providedwiththeNEBuilderHiFiDNAAssemblyCloningKitarerecommendedforuseforassembledproductsoflessthan15kb.ItisalsopossIBLetouseotherNEBcompetentE.colistrains,withtheexceptionofBL21,BL21(DE3),Lemo21(DE3),Nico(DE3),andSHuffle?.WhenusingcompetentE.colifromavendorotherthanNEB,wehavebeendecreasedrobustnessoftransformationwithhigh-fidelityDNAassembledproducts.Electroporation:Electroporationcanincreasetransformationefficiencybyseverallogs.WhenusingtheNEBuilderHiFIDNAAssemblyMasterMix,use1μloftheassembledproductforelectroporation,andplatemultipledilutions.ShouldyourequiretheuseofElectrocompetentcells,pleaseusethe"ElectrocompetentCellsTransformationProtocol"Biology:SomeDNAstructures,includinginvertedandtandemrepeats,areselectedagainstbyE.coli.SomerecombinantproteinsarenotwelltoleratedbyE.coliandcanresultinpoortransformationorsmallcolonies.
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