Description: NEB uilderHiFiDNAAssemblyCloningKitwasdevelopedtoimprovetheefficiencyandaccuracyofDNAassembly.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.
NEBuilderhasbeenusedinvariousapplicationsincluding:
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®5-alphaCompetentE.coli (HighEfficiency) -80 SOCOutgrowthMedium 4 1X NEBuilder®PositiveControl -20 2X pUC19Vector -20 0.05ng/μl
Notes: Uponarrival,storethekitcomponentsat–80°C.Beforeuse,thawandvortexthemastermixthoroughlyandkeeponice.Afterfirstuse,storetheHiFiDNAAssemblyMasterMix,SOCOutgrowthMediumandpositivecontrolsat–20°C.Storethecompetentcellsat–80°C.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.Itisalsoposs
IBL etouseotherNEBcompetentE.colistrains,withtheexceptionofBL21,BL21(DE3),Lemo21(DE3),Nico21(DE3),andSHuffle®.WhenusingcompetentE.colifromavendorotherthanNEB,wehaveseendecreasedrobustnessoftransformationwithhigh-fidelityDNAassembledproducts.Electroporation:Electroporationcanincreasetransformationefficiencybyseverallogs.WhenusingtheNEBuilderHiFiDNAAssemblyMasterMix,use1μloftheassembledproductforelectroporation,andplatemultipledilutions.ShouldyourequiretheuseofElectrocompetentcells,pleaseusethe"ElectrocompetentCellsTransformationProtocol"Biology:SomeDNAstructures,includinginvertedandtandemrepeats,areselectedagainstbyE.coli.SomerecombinantproteinsarenotwelltoleratedbyE.coliandcanresultinpoortransformationorsmallcolonies.