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当前位置: 首页 > 产品中心 > Difficult Cloning or BAC/Fosmid Cloning > Lucigen/CopyControl™ HTP Fosmid Library Production Kit/CCFOS059/1 Kit
公司介绍
美国Lucigen公司,自1998年成立至今,一直致力于生命科学领域相关科研产品的研究与开发,在分子生物学领域处于领导性地位。Lucigen公司主要开发各类用于基因克隆的试剂盒及相关产品,包括:CloneSmart®平端克隆试剂盒、BigEasy®线性克隆系统、pEZSeq™平端克隆试剂盒、ClonePlex™ AK文库构建试剂盒、DNA Terminator ®末端修复试剂盒、EconoTaq® DNA聚合酶及E.cloni感受态细胞等
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Lucigen/CopyControl™ HTP Fosmid Library Production Kit/CCFOS059/1 Kit

Lucigen/CopyControl™ HTP Fosmid Library Production Kit/CCFOS059/1 Kit
  • Lucigen/CopyControl™ HTP Fosmid Library Production Kit/CCFOS059/1 Kit
商品介绍

Efficientandimprovedsystemsforconstructinglibrariesofapproximately40kbclonesinbothstandardorhigh-throughputscreeningversions.

  • Formerly from EpicentrePrepareunbiasedfosmidlibrarieswithacompletekit,includingGELaseenzymeandMaxPlaxLamBDaPackagingExtracts
  • StABIlizecloneswithcopynumbercontrol
  • Optimizehigh-throughputend-sequencingresultswithCopyControlHTPFosmidLibraryProductionKit

Applications

  • Preparationofcompleteandunbiasedfosmidlibraries,fromanyBIOLOGicalsample,thatcanbemaintainedatsingle-copynumberandinducedtohigh-copynumberasneeded,usingtheCopyControl™AutoinductionSolution.
  • Constructionofmetagenomiclibrariesfrommicrobespresentinenvironmentalsamplessuchaswaterandsoil.

TheCopyControl™FosmidLibraryProductionKits*provideanefficientandimprovedmethodforconstructingalibraryofapproximately40kbclones.TheCopyControlpCC1FOS™VectorcontainsboththeE.coliF-factorsingle-copyoriginofreplicationandtheinducIBLehigh-copyoriV(Fig.1).CopyControlFosmidclonesaretypicallygrownatsinglecopytoensureinsertstabilityandsuccessfulcloningofencodedandexpressedtoxicproteinandunstableDNAsequences.TheCopyControlFosmidclonescanthenbeinducedupto50copiespercellimmediatelybeforeDNApurification.ThisstepgreatlyincreasesDNAyields,whilemaintainingthestabilityoftheplasmid.

TheCopyControlHTPFosmidLibrarycontainsthepCC2FOS™Vectorwhichisdesignedtooptimizeend-sequencingresults,especiallyinahigh-throughputsetting.Theprimercassette,engineeredinconjunctionwithAgencourtBioscienceCorporation,eliminateswastefulandexpensivevectorsequencereadsbyhavingthe3´endsoftheprimer-annealingsitesonlythreebasesfromthevector/insertjunction.Inaddition,theseven-basesequenceatthe3´endofeachprimerwasspecificallydesignedtominimizemisprimingtoanycontaminatingE.coliDNApresentaftertemplatepurification(Fig.2).

Thekitusesastrategyofcloningblunt-endedDNAfragmentsgeneratedbyrandomshearingoftheDNA,toproducemorecompleteandunbiasedgenomiclibrariesthancanbeobtainedbypartialrestrictionendonucleasedigests.GenomicDNAisfirstshearedintoapproximately40-kbfragments.TheshearedDNAisend-repairedtogenerateblunt,5´-phosphorylatedendsandthensize-selectedbyandrecoveredfromalow-melting-pointagarosegel.Finally,thesize-selectedDNAisligatedintothecloning-readyCopyControlpCC1FOSorpCC2FOSVector,packagedusingultra-highefficiencyMaxPlax™LambdaPackagingExtracts(>109pfu/µgDNA),includedinthekit,andplatedonthesuppliedTransforMax™EPI300™E.coli(Fig.3).

Figure1.CopyControl™Vectormap.TheCopyControlpCC1FOS™andpCC2FOS™VectorsforCopyControlFosmidlibraryproductionaresuppliedlinearizedattheEco72I(blunt)siteandthendephosphorylated.Thevectorisreadyforcloningend-repaired(blunt-end)genomicDNAofapproximately40kb.

Figure2.TheCopyControl™pCC2FOS™Vectorprimercassette.ThevectordiffersfromthepCC1FOSVectorbytheengineeringofanewprimercassettethateliminateswastefulvector-derivedsequencingreadsandminimizesthepotentialforprimingontheE.coligenome.

Benefits

  • CopyControlpCC1FOSandpCC2FOSVectorsaresuppliedCloning-Ready:linearized,dephosphorylated,purified,andreadyforligation.
  • NoneedforpartialrestrictionendonucleasedigestsorpulsefieldgelelectrophoresistopreparethegenomicDNAforcloning.
  • Maximizehigh-throughputend-sequenceresultsusingthepCC2FOSVector(Fig.4).
  • Clonescanbeinducedfromsinglecopyupto50copiespercell(Fig.5).SafelyobtainhigherDNAyieldswhilemaintainingthestabilityofsingle-copy-numberclones.
  • High-efficiencylambdapackagingeliminatesbackgroundandfalsepositives.
  • "Hands-off"autoinductionprotocolisperfectforhigh-throughputapplications.
  • FasterandeasierthanBACcloning.

Figure3(clicktoenlarge).OverviewoftheprocessforpreparingafosmidlibraryusingtheCopyControl™FosmidLibraryProductionKits.Oncethelibraryhasbeenprepared,individualclonescanbeculturedinsmallvolumeandinducedtomultiple-copynumberforhighyieldsofhigh-purityDNAforfingerprinting,sequencing,etc.,usingEpicentre'sDirectLysisFosmid96kitorFosmidMAX™DNAPurificationKit.

Figure4.TypicalsequencingresultsobtainedwiththepCC2FOS™ForwardPrimeronapCC2FOS™cloneat1/48xBigDye™dilution.SimilarresultswereobtainedwiththepCC2FOSReversePrimer(datanotshown).

Citations

  1. FEMSMicrobiolLett284(2008)28-34

*Coveredbyissuedand/orpendingpatents.

Figure5.CopyControl™Fosmidclonescanbeinducedupto50copiespercelltogreatlyincreaseDNAyield.HindIIIdigestsoffosmidDNAisolatedfromuninduced(–)andinduced(+)CopyControlclones.Digestscontainedone-third(8µl)ofthetotalsamplevolumeandwereanalyzedbyagarosegelelectrophoresis.LaneM,Kilobaseladder.

ORDERINFORMATION

CCFOS110producesupto10completeandunbiasedfosmidlibraries.Contents:CopyControl™pCC1FOS™FosmidVector,End-RepairEnzymeMix,End-Repair10XBuffer,dNTPMix,Fast-Link™DNALigase,Fast-Link™10XLigationBuffer,ATPSolution,GELase™EnzymePreparation,GELase™50XReactionBuffer,MaxPlax™LambdaPackagingExtracts,FosmidControlDNA,LigatedLambdaControlDNA,EPI300™PlatingStrain,ControlLambdaPlatingStrain,CopyControl™FosmidAutoinductionSolution.

CCFOS059producesupto10completeandunbiasedfosmidlibraries.Contents:CopyControl™pCC2FOS™FosmidVector,End-RepairEnzymeMix,End-Repair10XBuffer,dNTPMix,Fast-Link™DNALigase,Fast-Link™10XLigationBuffer,ATPSolution,GELase™EnzymePreparation,GELase™50XReactionBuffer,MaxPlax™LambdaPackagingExtracts,FosmidControlDNA,LigatedLambdaControlDNA,EPI300™PlatingStrain,ControlLambdaPlatingStrain,CopyControl™FosmidAutoinductionSolution.

CCIS125isa1,000Xconcentratedsolutionthathasbeenfiltersterilized.

品牌介绍

美国Lucigen公司,自1998年成立至今,一直致力于生命科学领域相关科研产品的研究与开发,在分子生物学领域处于领导性地位。Lucigen公司主要开发各类用于基因克隆的试剂盒及相关产品,包括:CloneSmart®平端克隆试剂盒、BigEasy®线性克隆系统、pEZSeq™平端克隆试剂盒、ClonePlex™ AK文库构建试剂盒、DNA Terminator ®末端修复试剂盒、EconoTaq® DNA聚合酶及E.cloni感受态细胞等。Lucigen公司凭借其独到的产品技术,过硬的产品质量,良好的产品服务赢得了全球广大用户的信赖。


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美国Lucigen公司,自1998年成立至今,一直致力于生命科学领域相关科研产品的研究与开发,在分子生物学领域处于领导性地位。Lucigen公司主要开发各类用于基因克隆的试剂盒及相关产品,包括:CloneSmart®平端克隆试剂盒、BigEasy®线性克隆系统、pEZSeq™平端克隆试剂盒、ClonePlex™ AK文库构建试剂盒、DNA Terminator ®末端修复试剂盒、EconoTaq® DNA聚合酶及E.cloni感受态细胞等。Lucigen公司凭借其独到的产品技术,过硬的产品质量,良好的产品服务赢得了全球广大用户的信赖。

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