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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1.如果在引物的5’引物引入了T7启动子,3‘引物也引入了polyT,PCR扩增出全长片段,回收纯化之后可以用来直接做体外转录么?为什么看到文献里都是先把这个PCR片段连接到载体里面,扩增之后提取出来线性化之后,再进行体外转录。是不是转录对模板的量有要求?
2.如果使用一些带有T7启动子序列的载体比如pBluescriptSK,在启动子序列和酶切位点之间还有多余的一段序列,这段序列也会被启动子转录,这一小段多余的序列是否会对接下来的转染以及病毒拯救带来影响?
3.有人在做反向遗传么,哪些载体可以用来进行体外转录?插入片段为7.5K。
2) 克隆技术使用使倾向于量繁殖现种群利用价值体,按自规律促进整种群优胜劣汰.意义说,克隆技术干扰自进化程.
3) 克隆技术种昂贵技术,需要量金钱物专业士参与,失败率非高.莉277实验唯.虽现发展更先进技术,功率能达2-3%.
4) 转基物提高疾病传染风险.例,产药物牛奶牛染病毒,种病毒能通牛奶染病
5) 克隆技术应用于体导致代遗传性状工控制.克隆技术引起争论核能否允许发育初期类胚胎进行遗传操作.伦理家所能接受.
6) 克隆技术用创造超,或拥健壮体格却智力低.且,克隆技术能够类效运用,男性失遗传意义.
7) 克隆技术家庭关系带影响巨.由父亲DNA克隆孩看作父亲双胞胎兄弟,延迟几十已.难设想,发现自另外完全复制品,(或)受
1、RNA干扰基因蛋白的表达的过程是瞬间的还是一步步的,需要一段时间才能使目的基因沉默。
2、关于siRNA的合成,有好多种,但文献报道的一般是化学合成的和通过构建质粒表达载体完成的,但据俺了解,体外转录和RNA酶III酶切从时间上和价格上都比这两种方法好,为何这么多研究者青睐这两种方法呢
3、俺打算用此方法研究某个基因的功能,想先进行体外实验,然后再进行在体实验,请教各位,用哪种方法比较好?
4、如果用质粒的话,究竟用哪种质粒好呢?听说质粒比较昂贵,看到好多文献所用质粒都是惠赠的,怎么才能通过惠赠的方式获得质粒载体呢
或许俺提的这些问题过于低级,恳切希望各位高手予以解答,不才万分感激!
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