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主要产品：NEB、内切酶、基因表达、RNAi DNA修饰酶、各种载体、 Marker 、引物、测序 等等
℡ 4000-520-616
℡ 4000-520-616
NEB/Q5® Site-Directed Mutagenesis Kit (Without Competent Cells)/E0552S/10 reactions
产品编号：E0552S-10reactions
市  场 价：¥2620.00
场      地：美国(厂家直采)
联系QQ：1570468124
电话号码：4000-520-616
邮      箱： info@ebiomall.com
美  元  价：$131.00
品      牌： NEB
公      司：New England Biolabs
公司分类：
NEB/Q5® Site-Directed Mutagenesis Kit (Without Competent Cells)/E0552S/10 reactions
商品介绍

Description:

TheQ5Site-DirectedMutagenesisKit(WithoutCompetentCells)enablesrapid,site-specificmutagenesisofdouble-strandedplasmidDNAinlessthan2hours(Figure1).ThekitutilizestherobustQ5HotStartHigh-FidelityDNAPolymerasealongwithcustommutagenicprimerstocreateinsertions,deletionsandsubstitutionsinawidevarietyofplasmids.AfterPCR,theamplifiedmaterialisaddeddirectlytoauniqueKinase-Ligase-DpnI(KLD)enzymemixforrapid(5minutes),roomtemperaturecircularizationandtemplateremoval(Figure2).Transformationintohigh-efficiencychemically-competentE.coli,notsupplied,ensuresrobustresultswithplasmidsuptoatleast20kbinlength.

Figure1:Site-specificmutagenesisproceedsinlessthan2hours.Figure 1: Site-specific mutagenesis proceeds in less than 2 hours.

Theuseofamastermix,auniquemulti-enzymeKLDenzymemix,andafastpolymeraseensuresthat,formost plasmids,themutagenesisreactioniscompleteinlessthantwohours.
   
Figure2:Q5Site-DirectedMutagenesisOverview.Figure 2: Q5 Site-Directed Mutagenesis Overview.

Thiskitisdesignedforrapidandefficientincorporationofinsertions,deletionsandsubstitutionsintodoublestranded plasmidDNA.Thefirststepisanexponentialamplificationusingstandardprimersandamastermix fomulationofQ5HotStartHigh-FidelityDNAPolymerase.Thesecondstepinvolvesincubationwithaunique enzymemixcontainingakinase,aligaseandDpnI.Together,theseenzymesallowforrapidcircularizationofthe PCRproductandremovalofthetemplateDNA.Thelaststepisahigh-efficiencytransformationintochemicallycompetent cells(notprovided).
Figure3:PrimerDesignforQ5Site-DirectedMutagenesisFigure 3: Primer Design for Q5 Site-Directed Mutagenesis

Substitutions,deletionsandinsertionsareincorporatedintoplasmidDNAthroughtheuseofspecificallydesigned forward(black)andreverse(red)primers.Unlikekitsthatrelyonlinearamplification,primersdesigned fortheQ5Site-DirectedMutagenesisKitshouldnotoverlaptoensurethatthebenefitsof exponentialamplificationarerealized.A)Substitutionsarecreatedbyincorporatingthedesirednucleotide change(s)(denotedby*)inthecenteroftheforwardprimer,includingatleast10complementarynucleotideson the3´sideofthemutation(s).Thereverseprimerisdesignedsothatthe5´endsofthetwoprimersannealbackto-back.B)Deletionsareengineeredbydesigningstandard,non-mutagenicforwardandreverseprimersthatflank theregiontobedeleted.C)Insertionslessthanorequalto6nucleotidesareincorporatedintothe5´endofthe forwardprimerwhilethereverseprimerannealsback-to-backwiththe5´endofthecomplementaryregionofthe forwardprimer.D)Largerinsertionscanbecreatedbyincorporatinghalfofthedesiredinsertionintothe5´ends ofbothprimers.Themaximumsizeoftheinsertionislargelydictatedbyoligonucleotidesynthesislimitations.
Figure4:NEB’sQ5SDMKitdelivershighertransformationefficiencythanAgilent’sQuikChange®SDMKit
Q5 Graph
Resultsfromasubstitutionreaction(4nt)usingtheback-to-backControlSDMPrimerMixandControlSDMPlasmid(6.7kb)areshown,alongwithresultsfroma12ntdeletionexperiment(5.8kbplasmid)andan18ntinsertionexperiment(7.0kbplasmid).Inallthreecases,over90%oftheresultantcolonieshadincorporatedthedesiredmutation(s).Resultsarenormalizedtototaltransformantsifcellswerenotdilutedpriortoplating.Forcomparison,thesamesubstitutionreaction(4nt)wasperformedwiththeQuikChangeLightningSite-DirectedMutagenesisKit(Agilent)followingAgilent’sprotocolandusingAgilent’sprimerdesigntooltodesignoverlappingprimers.

*NotethattheQuikChangekitdoesnotaccommodatedeletionsandinsertionsofthissize,sonocomparisoncouldbemadefortheseexperiments.

KitComponents

Thefollowingreagentsaresuppliedwiththisproduct:

Storeat(°C)Concentration
Q5®HotStartHigh-Fidelity2XMasterMix-202X
KLDEnzymeMix-2010X
KLDReactionBuffer-202X
ControlSDMPrimerMix-2010μM
ControlSDMPlasmid-205μg/ml

Notes:

StorageNote:TheQ5Site-DirectedMutagenesisKit(WithoutCompetentCells)isstableat–20°Cfortwoyears.Forflexibility,themutagenesisreagentsandcontrolreactionsaresuppliedwithoutcompetentcellssothatanychemically-competentE.colicellssuitableforcloningmaybeused.
品牌介绍

NEB公司成立于二十世纪七十年代中期，拥有众多经验丰富的科学家，是生产生命科学试剂的领导者。目前，NEB为基因组研究提供最齐全的重组酶和天然酶，并且公司业务范围已延伸至蛋白质组学和药品开发领域。回顾三十余年来的历程，NEB公司作为先驱公司之一，为促进生物科技工业的发展做出了巨大的贡献。NEB美国总部乔 迁新址后拥有最尖端的设备，有一座现代化的发酵中心及设备齐全的实验室，这些实验室主要用于产品生产、质量监控、 产品开发和基础科研之用。作为首批以商业规模生产限制性内切酶的公司之一，NEB一直专注于内切酶的研究，并保持 业内领先水平。NEB公司一贯坚持以科学为本的原则，公司生产的试剂因其高质量、高性价比享誉世界。NEB（北京）有限公司 New England Biolabs (Beijing) LTD. NEB（北京）有限公司New England Biolabs (Beijing) LTD. 为美国New England Biolabs, Inc.在华投资兴建的独资子公司，负责处理其在中国内地及香港地区的全部业务，同时承担部分新产品的研发工作。纽英伦生物技术（北京）有限公司成立于2001年11月，为中国生命科学研究者提供一流的产品和专业的技术服务，促进国内外生命科学学术交流，与中国科研工作者一起将中国推入到二十一世纪生命科学的前沿中去。

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