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  • EXERCICIOS Cloning PCR PRODUCTS Exercício PDF

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    PCR products that possess single adenylate 3' overhangs at each end can be cloned into a vector. Restriction endonuclease sites are added to the 3' termini of an amplified fragment. The resultant DNA fragment with cohesive ends will anneal to complementary sequences on a prepared cloning vector.

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    PCR products that possess single adenylate 3' overhangs at each end can be cloned into a vector. Restriction endonuclease sites are added to the 3' termini of an amplified fragment. The resultant DNA fragment with cohesive ends will anneal to complementary sequences on a prepared cloning vector.

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    EXERCICIOS Cloning PCR PRODUCTS Exercício PDF

    Diunggah oleh

    FCiências
    PCR products that possess single adenylate 3' overhangs at each end can be cloned into a vector. Restriction endonuclease sites are added to the 3' termini of an amplified fragment. The resultant DNA fragment with cohesive ends will anneal to complementary sequences on a prepared cloning vector.

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    Cloning PCRPRODUCTS

    TherearethreecommonstrategiesforcloningPCRproductsintoplasmidvectors. http://www.mfa.od.ua/page152.htm

    1.BluntEndLigation ThefirstapproachinvolvescloningPCR productsintoaplasmidthathasbeen digestedtoproducebluntends. Pfu DNAPolymerase,forhighfidelity PCRandprimerextensionreactions, PCRcloning,andgenerating bluntend amplificationproduct

    2.TACloning Clark(2)describedtemplateindependent terminaltransferase activityofTaq DNA polymerase inwhichtheenzymeaddsasingle nucleotidetothe3'terminiofanamplified fragment. Althoughthisactivitymayresultinthe additionofanyofthefournucleotidebases, thereisastrongbiasfortheadditionof adenylate. PCRproductsthatpossesssingleadenylate 3' overhangsateachendcanbeclonedintoa vectorpossessingsinglethymidine3' overhangs andforthisreason,theprocessis frequentlyreferredtoas"TAcloning"andthe acceptingvectorasa"Tvector."

    http://www.genomics.agilent.com/article.jsp?pageId=1268

    http://mvz.berkeley.edu/egl/resources/product%20inserts/TAcloning.pdf

    http://www.lifetechnologies.com/order/catalog/product/K203001

    3.AdditionofRestrictionEndonucleaseSitesinto PCRProduct Thesecondcloningstrategyutilizesoligonucleotide primersdesignedtoincluderestrictionenzyme recognitionsitesneartheir5'termini. Followingamplification,theresultantproductis isolatedbyprecipitationandcleaved bythe appropriaterestrictionendonucleases,resultingin aDNAfragmentwithcohesiveendsthatwill annealtocomplementarysequencesona preparedcloningvector.

    Molecular Cloning: A Laboratory Manual, Volume 1


    Joseph Sambrook, David William Russell Cold Spring Harbor Laboratory Press, 2001 - 2344 pginas

    EXERCCIO1
    AenzimaTaq DNApolimeraseintroduzumAextraemcadaextremidade3doproduto amplificado.Estacaractersticapermite(assinalaraopocorrecta) a)aclonagemdireccional b) aclonagememvectorescomumTadicionala5decadaextremidadedolocalde insero c) aclonagememvectorescomumTadicionala3decadaextremidadedolocalde insero d) ousoobrigatriodeumacinase nucleotdica parafosforilar oprodutodePCR e)permiteaclonagememlocaisdeinseroblunt

    EXERCCIO2 Apresenadesequnciasdereconhecimentodiferentesnasextremidadesdeum amplificado,porexemploparaEcoRI (5G/AATTC3)eHindIII (5A/AGCTT3)(V/F) __permiteaclonagemdireccionaldoprodutodePCR __permiteaclonagemembluntdoprodutodePCR __asextremidadescriadaspelasreferidasenzimassocompatveis,logoainsero novectorpodeocorreremqualquerdireco __antesdainseronovector,oprodutodePCRdeversercortadoexclusivamente comEcoRI __antesdainseronovectoroprodutodePCRdeversersubmetidoadigesto duplacomHindIII eEcoRI

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