- Convenient,pre-processedvectorseliminatetheneedfordigestion,gelpurification,anddephosphorylation.
- TranscriptionterminatorsflankingcloningsitestABIlizeotherwisetoxicinserts.
- Choiceofcopynumberandantibioticresistance.
- OptimizedforusewithE.cloni®10Gelectrocompetentandchemicallycompetentcells(soldseparately)
Clonequicklyandfacilitatedownstreamapplications
Lucigen’suniquecloningkitswithpre-processedvectorsandhighlycompetentcellseliminatehoursoftediouspreparationandenableefficientcloning.Ligationcanbecompletedinaslittleas30minuteswithnoclean-upneeded;onlyabriefheatdenaturationisnecessarybeforetransformation.Downstreammanipulation(e.g,mutagenesis,subsequentinsertaddition)isfacilitatedbytheminimalvectorsize(1.7-2.0kb).
StabilizedInsertsandGap-FreeCloning
Allcloningkitsincludethetranscription-andtranslation-freepSMART®vectors(Figure1),whicheliminatemanyoftheproblemsassociatedwithcloningrecalcitrantDNAinconventionalplasmidvectors.Conventionalcloningvectorscontainpromoters(e.g.,lacZpromoter)thatconstitutivelytranscribetheinsertsequence.Thistranscriptionandcoupledtranslationcandestabilizeinsertsthatcontaincodingregions,strongpromoters,shortrepeats,orincompatIBLesecondarystructures.ThecloningsiteofpSMARTvectorsdoesnotincludealaczsequence.Inaddition,strongtranscriptionterminatorsflankthecloningsitetoblockspurioustranscriptionfromthevectorandinsert-driventranscriptionintothevector.LowcopynumberversionsofpSMARTfurtherstabilizesequencesthataredifficulttomaintainintypicalvectors.
NoBackgroundProblems
ThepSMARTvectorsaresuppliedpre-digested,withblunt,dephosphorylatedends,andarequalifiedtoproduce99.5%recombinantclonesintypicalexperiments.Theultra-lowbackgroundofemptyvector(lessthan0.5%)eliminatestheneedtoscreenforrecombinants,removestheuncertaintyoffalsenegatives(lightbluepUCcolonies)andfalsepositives(whitecoloniesthatlackinserts),andenableslibraryconstructionfromnanogramamountsofDNA.
Figure1.Highcopy(HC)andlowcopy(LC)versionsofthepSMARTtranscription-freecloningvectors.
ConvenientSuccess
EfficientlyobtainyourcloneorcreateyourgenomicorCDNAlibrary—theCloneSmartCloningKitseliminatetediouspreparationofvectorsandcompetentcells,aswellastime-consumingQCtestingexperiments.Kitsincludeoptimizedreagents,ligation-readyvector(nopost-ligationcleanupsteprequired),detailedinstructions,andtrouble-shootingguidestosimplifycloningandsequencing.HighlyefficientE.cloni®10Gelectrocompetentcells(upto>4×1010cfu/µg)orchemicallycompetentcells(>1×109cfu/µg)areavailableforsaleseparately.
ORDERINFORMATION
CloneSmartBluntCloningKitscontain:VectorPremix,CloneSmartDNALigase,PositiveControlInsertDNA,SequencingPrimers,andacompleteprotocol.KitscanbeusedwithanyE.colichemicallycompetentorelectrocompetentcellsbuthavebeenoptimizedforusewithofE.cloni®ElectrocompetentCellsandChemicallyCompetentCells.ebiomall.com
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采用6孔板接毒后,铺上低溶解度琼脂糖,第二天就出来了这些病变,正常细胞没有出现这种现象。
按说空斑应该出现多个,但我的六孔板中一个空最多也就出现了2个空斑,也不像文献报道的那样规则,而且很大!
由于没有做梯度稀释,会不会是接毒量过大造成的啊?请大侠指点,万分感谢!
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.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。
般基克隆物体直接取基包括面几步骤
提取植物或者物基组DNA(试剂盒)————做PCR需要基扩增(顺利2搞定)——扩增基做酶切——基连接想插入载体面(顺利1-2)——鉴定克隆基
步骤都商品化试剂或者试剂盒 要思路清晰做起快 1-2周能完