Description: (USACustomers:Pleasenoteyouwillreceivetwoseparateboxeswhenyouorderthisproduct;oneboxwillcontainthecompetentcellsondryice;theotherboxwillcontain
NEB uilderHiFiDNAAssemblyMasterMixonwetice.)
NEBrecommendsNEB10-betaCompetent
E.coli (HighEfficiency,NEB#C3019)forassemblieslargerthan15kb.NEBuilderHiFiDNAAssemblyBundleforLargeFragmentsoffersadiscountforpurchasingthesecompetentcellswithNEBuilderHiFiDNAAssemblyMasterMix.
NEBuilderHiFiDNAAssemblywasdevelopedtoimprovetheefficiencyandaccuracyofDNAassembly.ThismethodallowsforseamlessassemblyofmultipleDNAfragments,regardlessoffragmentlengthorendcompatibility.Thismethodhasbeenusedtoassembleeithersingle-strandedoligonucleotidesordifferentsizesofDNAfragmentswithvariedoverlaps(15-80bp).Ithasutilityforthesynthetic
BIOLOG ycommunity,aswellasthoseinterestedinone-stepcloningofmultiplefragmentsduetoitseaseofuse,flexibilityandsimplemaster-mixformat.Thereactionincludesdifferentenzymesthatworktogetherinthesamebuffer(seeFigure1):
Theexonucleasecreatessingle-stranded3´overhangsthatfacilitatetheannealingoffragmentsthatsharecomplementarityatoneend(theoverlapregion) Thepolymerasefillsingapswithineachannealedfragment TheDNAligasesealsnicksintheassembledDNA Theendresultisadouble-strandedfullysealedDNAmoleculethatcanserveastemplateforPCR,RCAoravarietyofothermolecularbiologyapplications,includingdirecttransformationof
Ecoli . NEBuilderHiFiDNAAssemblyCloningKitissuppliedwithNEB5-alphaHighEfficiencyCompetent
E.coli .
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:OverviewoftheNEBuilderHiFiDNAAssemblyMethod Figure2:NEBuilderHiFiDNAAssemblyoffersimprovedefficiencyandaccuracyover NEBGibsonAssembly Reactionsweresetupina2-and6-fragmentassemblyreactionaccordingtorecommendedreactionconditions.NEBuilderHiFiDNAAssemblyresultsinlargernumbersofcoloniesoverNEBGibsonAssembly,forboth2-and6-fragmentassemblies. ViewadditionalperformancedatacomparedtoNEBGibsonAssembly Figure3:NEBuilderHiFidelivershighercolonyyieldthanIn-FusionHD Two-fragmentreactionsweresetupusingthepositivecontrolfromtheIn-FusionHDCloningKit(ClontechTakaraBioUSA,Inc),accordingtorecommendedprotocols.2μlofassemblyreactionwastransformedintosuppliedcompetentcells.1/50ofoutgrowthwasspreadonanApR plate. ViewadditionalperformancedatacomparedtoIn-FusionHD ComparisonofDNAAssemblyReactionTypes
KitComponents Thefollowingreagentsaresuppliedwiththisproduct:
Storeat(°C) Concentration NEBuilder®High-FidelityMasterMix -20 NEB®10-betaCompetentE.coli (HighEfficiency) -80 SOCOutgrowthMedium 4 1X NEBuilder®PositiveControl -20 2X pUC19Vector -20 0.05ng/μl
Notes: Youwillreceive3separateproductswhenyouorderthisbundle:2X(NEB#E2621S)and1X(NEB#C3019H)(competentcellswillarriveinaseparateboxcontainingdryice).StoretheHiFiDNAAssemblyMasterMixandpositivecontrolsat-20°C.StoretheSOCOutgrowthMediumat4°C.Storethecompetentcellsat-80°C.ToensurethesuccessfulassemblyandsubsequenttransformationofassembledDNAs,NEBrecommendsthefollowing:DNA:PCRproductpurificationisnotnecessaryifthetotalvolumeofallPCRproductsis20%orlessoftheassemblyreactionvolume.HighervolumesofPCRproductsmayreducetheefficiencyofhigh-fidelityDNAassemblyandtransformationduetotheelevatedcarryoveramountsofPCRreactionbufferandunusedprimerspresentinthePCRproduct.ColumnpurificationofPCRproductsmayincreasetheefficiencyofbothhigh-fidelityDNAassemblyandtransformationby2-10foldandishighlyrecommendedwhenperformingassembliesofthreeormorePCRfragmentsorassemblinglongerthan5kbfragments.PurifiedDNAforassemblycanbedissolvedinddH2O(Milli-Q®waterorequivalentispreferable),TEorotherdilutionbuffers.Insert:Whendirectlyassemblingfragmentsintoacloningvector,theconcentrationofassemblyfragmentsshouldbeatleast2timeshigherthantheconcentrationofvector.Forassemblyof4ormorefragmentsintoavector,werecommendusinganequimolarratiooffragments.Biology:SomeDNAstructures,includinginvertedandtandemrepeats,areselectedagainstbyE.coli.SomerecombinantproteinsarenotwelltoleratedbyE.coliandcanresultinpoortransformationorsmallcolonies.