Efficientandimprovedsystemsforconstructinglibrariesofapproximately40kbclonesinbothstandardorhigh-throughputscreeningversions.
Applications
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
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Figure3(clicktoenlarge).OverviewoftheprocessforpreparingafosmidlibraryusingtheCopyControl™FosmidLibraryProductionKits.Oncethelibraryhasbeenprepared,individualclonescanbeculturedinsmallvolumeandinducedtomultiple-copynumberforhighyieldsofhigh-purityDNAforfingerprinting,sequencing,etc.,usingEpicentre'sDirectLysisFosmid96kitorFosmidMAX™DNAPurificationKit. Figure4.TypicalsequencingresultsobtainedwiththepCC2FOS™ForwardPrimeronapCC2FOS™cloneat1/48xBigDye™dilution.SimilarresultswereobtainedwiththepCC2FOSReversePrimer(datanotshown). Citations
*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.
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现在小弟碰见一个难题,我想克隆一个基因,但是我在NCBI一检索,发现别人只发表了这个基因的DNA全长,但是,CDNA没有发表,也没有发表文章,想请问下,在不知道CDNA的情况下,请问我应该如何做这个基因的克隆???(全基因组也不知道,其他生物没有这种基因)
详见:http://zhidao.baidu.com/question/1831844001436785260.html?fr=iks&word=%B2%A1%B6%BE%B5%C4%B8%B4%D6%C6%D6%DC%C6%DA%B5%C4%B9%FD%B3%CC%3F&ie=gbk
SeamlessCloning(无缝克隆)技术提供了一种新的、高效快速的基因克隆方法,只要插入的DNA片段末端与载体末端具有15-25个重叠碱基序列就可以在载体的任意位点完成克隆重组。
产品特点:
1.30分钟可以将一个或者多个长、短PCR扩增片段(平端、A端均可)插入载体。
2.不受载体和插入片段酶切位点的可用性和平端/粘性末端的限制,可以在任意位点进行克隆。
3.无缝克隆,插入点不会引入不需要的碱基序列。
4.高效、准确,阳性率>95%。
无缝克隆技术严格意义不是新技术,类似于乔布斯天才的集成了现成的电子元器件造出Iphone,开创了智能手机新时代一样。美国的吉布斯在2009年首次用一个新颖的思路,将3种现有的商品化的酶组合起来,达到了无缝克隆的效果,开创了无缝克隆新时代。因此,您也可以按照吉布斯的思路亲自组装无缝克隆试剂盒,领略天才的思路。当然你也可以通过购买北京艾德莱(aidlab)的无缝克隆试剂盒CV1201SeamlessAssemblyandCloningkit,体会无缝克隆的妙处。
原理说明:紫红色和绿色2个片段为需要连接的双链DNA片段,它们末端有相同的15-25个重叠序列(黑色),首先T5exonuclease核酸外切酶切去5’端的一些碱基,形成3’端突出的单链,3’端单链互补退火;然后Phusion高保真聚合酶补上两条单链之间的缺口(gaps);最后TaqDNAligase将相邻的单链裂口(nicks)连接补齐。
以上用到的三种酶NEB公司全部有商品化销售,因此,您可以DIY自己的无缝克隆试剂盒。但是艾德莱生物也提醒您,如果您不能自己表达纯化生产这些酶降低成本,那么购买这些商品化酶DIY也会比较昂贵,不是发烧友,还是购买艾德莱生物(aidlab)帮您精心准备的CV1201SeamlessAssemblyandCloningkit。http://www.aidlab.cn/products1.asp?anclassid=91
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