- Repairshearedorrestriction-digestedDNAends
- Increasecloningefficiencyasmuchasa500%
- Optimizedsystemforconsistent&reliableresults
RepairDNAends
Double-strandedDNAfragmentsforlibraryconstructionorlarge-scalesequencingareoftengeneratedbymechanicallyshearinglarger(genomic)DNA,aprocessthatprimarilyleavesunevenends.LargeDNAmayalsobecutwithrestrictionenzymestogeneratesmallerfragmentsforsubcloning.ShearedorrestrictedDNAfragmentsmustbe“end-repaired”beforeligationintoblunt-endcloningvectors(e.g.,Lucigen’sCloneSmart®,CopyRight®,orBigEasy®systems).
TheDNATerminatorEndRepairKitcreatesblunt,5'phosphorylatedendsonanytypeofshearedorrestriction-digestedDNAfragment,ensuringthehighestpossIBLecloningefficiency(Figure1).
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Figure1.EffectofDNAshearingandend-repairmethodsonlibraryconstructionefficiency.Shotgunlibrarieswereconstructedusing250ngoflamBDaDNAshearedbysonication(Son)orbytheGeneMachines®HydroShear™device(HS).ShearedDNAwasrepairedwithT4DNApolymeraseandKlenowfragment(TK)orDNATerminatorEndRepairKit(DT)andclonedusingLucigen’sCloneSmart®system.Valuesarecolonyformingunits(cfu)perlibrary. |
