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Epigentek/EpiNext DNA Library Preparation Kit (Illumina)/P-1051-24/24 reactions

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InputType:DNAResearchArea:NextGenSequencingTargetApplication:LibraryConstructionVesselFormat:Columns/Tubes100%Guarantee:6monthsProductOverviewTheEpiNext™ DNALibraryPreparationKit (Illumina)isacompletesetofoptimizedreagentstocarryoutasuccessfulDNAlibrarypreparation.ThekitissuitableforpreparingaDNAlibraryfornextgenerationsequencingapplicationsusinganIlluminasequencer,whichincludesgenomicDNA-seq,ChIP-seq,MeDIP/hMeDIP-seq,bisulfite-seq,andtargetedre-sequencing.Theoptimizedprotocolandcomponentsofthekitallowbothnon-barcoded(singleplexed)andbarcoded(multiplexed)DNAlibrariestobeconstructedquicklywithreducedbias.Thekithasthefollowingadvantages:Fastandstreamlinedprocedure-TheprocedurefromfragmentedDNAtosizeselectionislessthan2h30min. Onlyoneclean-upbetweeneachstep,therebysavingtimeandpreventinghandlingerrors,aswellaslossofvaluablesamples.Gel-freesizeselectionfurtherreducesthepreparationtime.HighlyConvenient-ThekitcontainsallrequiredcomponentsforeachstepofDNAlibrarypreparation,whicharesufficientforendrepair,dAtailing,ligation,clean-up,sizeselectionandlibraryamplification,therebyallowingthelibrarypreparationtobestreamlinedwiththemostreliableandconsistentresults. Minimizedbias-UltraHiFiamplificationandanoptionalPCR-freestepenabletheusertoachievereproducIBLyhighyieldsofDNAlibrarywithminimalsequencebiasandlowerrorrates.Flexibility-Canbeusedforbothnon-barcoded(singleplexed)andbarcoded(multiplexed)DNAlibrarypreparation. UsesvariousdsDNAincludingfragmenteddsDNAisolatedfromvarioustissueorcellsamples,dsDNAenrichedfromChIPreactions,MeDIP/hMeDIPreactions,orexoncapture.BroadrangeofinputDNAfrom10ngto1µg.PCR-freelibrarypreparationcanbeperformedwithuseof500ngormoreinputDNA.BackgroundInformationDNAlibrarypreparationisacriticalstepfornextgenerationsequencing(NGS).TogenerateaccuratesequencingdataforNGS,thepreparedlibraryDNAshouldbesufficientinyieldandofhighquality.Also,asNGStechnologyiscontinuouslyimproving,DNAlibrarypreparationisrequiredtobeoptimizedaccordingly. Mostofthecurrentlyusedmethodsareunfortunatelytime-consuming,expensive,andinconvenient.SomeofthemethodsarerelativelyquickbycombiningendrepairanddAtailingorevenligationinone-step,buthavebeenshowntogeneratesignificantGtailingorformconcatmersattheligationsteporhavehighinsertionbias.ThesesidereactionseventuallyresultinthepreparedDNAlibrarybeinglessefficientandinaccurate.AnidealDNAlibrarypreparationmethodshouldbebalancedinspeed,convenience,smallsample-suitABIlity,cost-effectiveness,andaccuracy.Toaddressthisissue,EpigentekofferstheEpiNextDNALibraryPreparationKit.Principle&ProcedureThiskitincluldesallreagentsrequiredateachsteptocarryoutasuccessfulDNAlibrarypreparation.Inthelibrarypreparation,DNAisfirstfragmentedtotheappropriatesize(about300bppeaksize).TheendrepairoftheDNAfragmentsisperformedandanA-overhangisaddedatthe3"-endofeachstrand. Adaptorsarethenligatedtobothendsoftheendrepaired/dAtailedDNAfragmentsforamplificationandsequencing.FragmentsarethensizeselectedandpurifiedwithMQbeads,whichallowsforquickandprecisesizeselectionofDNA.Size-selectedDNAfragmentsarethenamplifiedwithahigh-fidelityPCRMixwhichensuresmaximumyieldsfromminimumamountsofstartingmaterialandprovideshighlyaccurateamplificationoflibraryDNAwithlowerrorratesandminimumbias. StartingMaterialsStartingmaterialscanincludefragmenteddsDNAisolatedfromvarioustissueorcellsamples,dsDNAenrichedfromChIPreactions,MeDIP/hMeDIPreaction,orexoncapture.DNAshouldberelativelyfreeofRNAsincelargefractionsofRNAwillimpairendrepairanddAtailing,resultinginreducedligationcapabilities.InputamountofDNAcanbefrom5ngto1µg.Foroptimalpreparation,theinputamountshouldbe100ngto200ng.Foramplification-free,500ngormoreisneeded. Fig.1. WorkflowoftheEpiNext™DNALibraryPreparationKit(Illumina).Fig.2. Sizedistributionoflibraryfragments:humanplacentaDNAwasshearedto210bpspeaksizeand20ngofshearedDNAwasusedforDNAlibrarypreparationusingtheEpiNext™DNALibraryPreparationKit(Illumina).ProductComponents10XEndRepairBuffer*EndRepairEnzymeMix*10XdA-TailingBuffer*KlenowFragment(3’-5’exo-)*2XLigationBuffer*T4DNALigase*Adaptors(50µM)*MQBindingBeads*2XHiFiPCRMasterMix*PrimerU(10µM)*PrimerI(10µM)*ElutionBuffer*UserGuide*Spinthesolutiondowntothebottompriortouse.

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