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DigestcontaminatinglinearDNAwithoutdigestingnickedorclosed-circulardsDNAorsupercoiledDNA(plasmids,fosmids,etc.)Plasmid-Safe™ATP-DependentDNaseselectivelyremovescontaminatingbacterialchromosomalDNAfromplasmid,cosmid,fosmid,andBACclonesorvectorpreparations.SuchpreparationsarefrequentlycontaminatedwithfragmentsofbacterialgenomicDNAgeneratedduringalkalinelysis.Otherpurificationoptions,suchasspin-columnsorevenCsClcentrifugation,donoteffectivelyremovethesecontaminantsandrequirefurtherpurificationsteps.ContaminatingDNAfragmentsleftbehindbythesemethodsultimatelycanbecomeligatedintoacloningvector,resultinginfalsepositivesandhighbackgrounds,orerroneoussequencedata. Plasmid-SafeATP-DependentDNasedigestslineardsDNAtodeoxynucleotidesatslightlyalkalinepHand,withlowerefficiency,closed-circularandlinearssDNA.Theenzymehasnoactivityonnickedorclosed-circulardsDNAorsupercoiledDNA.Therefore,Plasmid-SafeDNaseisidealasthefinalpurificationstepforplasmid,cosmid,fosmid,andBACvectorandclonepreparations. |  | Figure1.Plasmid-Safe™ATP-DependentDNaseremovescontaminatinggenomicDNAfromplasmidpreps.Lane1,3µgofSma I-digestedbacterialchromosomalDNA;lane2,500ngofuncutplasmidDNA;lane3,mixtureof3 µgofdigestedbacterialchromosomalDNAand500ngofuncutplasmidbeforePlasmid-SafeDNasetreatment;lane4,mixtureofchromosomalDNAandplasmidDNAafterPlasmid-SafeDNasetreatment(incubationwithPlasmid-SafeDNasefor30minutesat37°C);laneM,Kilobaseladder. |
Benefits
UnitDefinition:OneunitofPlasmid-SafeDNaseconverts1nmolofdeoxynucleotidesinlinearT7DNAintoanacid-solubleformin30 minutesat37°Cunderstandardassayconditions.Threeunitswilldigest1µgofDNAin30minutesat37°C.
StorageBuffer:50%glycerolcontaining50mMTris-HCl(pH7.5),0.1MNaCl,0.1mMEDTA,1mMDTT,and0.1%Triton®X-100.
Plasmid-Safe10XReactionBuffer:330mMTris-acetate(pH7.5),660mMpotassiumacetate,100mMmagnesiumacetate,and5.0 mMDTT.ATPmustbeaddedtoafinalconcentrationof1mMinthe1XBuffer.
QualityControl:Plasmid-SafeDNaseisfreeofdetectableRNaseanddouble-stranded,DNA-specificendonucleaseactivities.
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