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  • Plasmid Modification by Annealed Oligo Cloning

    Enviado por

    Vempati Rahul Kumar
    155 visualizações4 páginas
    This document describes a method for modifying plasmids using annealed oligonucleotide cloning. The method involves designing overlapping oligos containing the desired DNA sequence, annealing the oligos, digesting and purifying the target plasmid, ligating the annealed oligos into the plasmid, and transforming the ligation reaction into bacteria. This technique can be used to add short DNA sequences like restriction sites or tags to plasmids.

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    This document describes a method for modifying plasmids using annealed oligonucleotide cloning. The method involves designing overlapping oligos containing the desired DNA sequence, annealing the oligos, digesting and purifying the target plasmid, ligating the annealed oligos into the plasmid, and transforming the ligation reaction into bacteria. This technique can be used to add short DNA sequences like restriction sites or tags to plasmids.

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    155 visualizações4 páginas

    Plasmid Modification by Annealed Oligo Cloning

    Enviado por

    Vempati Rahul Kumar
    This document describes a method for modifying plasmids using annealed oligonucleotide cloning. The method involves designing overlapping oligos containing the desired DNA sequence, annealing the oligos, digesting and purifying the target plasmid, ligating the annealed oligos into the plasmid, and transforming the ligation reaction into bacteria. This technique can be used to add short DNA sequences like restriction sites or tags to plasmids.

    Direitos autorais:

    © All Rights Reserved
    Baixe no formato PDF, TXT ou leia online no Scribd
    Sinalizar o conteúdo como inadequado
    de 4

    11/12/2016

    Addgene:PlasmidModificationbyAnnealedOligoCloning(withProtocols)

    PlasmidModificationbyAnnealedOligoCloning
    Summary

    Youmayalsolike...

    OligooverlapcloningcanbeusedanytimeyouneedtoaddashortstretchofDNAtoaplasmid,suchas:
    RestrictionDigestof
    PlasmidDNA

    ChangingaMultipleCloningSite(MCS)
    Addingshorttagsex:HAorFlag
    CloningshRNAs

    DNALigation
    BacterialTransformation

    Background
    ForthepurposesofthistutorialwewilldiscusshowtoaddnewrestrictionsitestotheMCSofanemptyvector.
    However,thefollowingtechniquecanbeusedjustaseasilytoaddshorttagsorshRNAstoanyvector(the
    procedurewillsimplydifferintermsofprimerdesign).

    Let'sassumethatyourfavoritevectorhasarelativelylimitedMCS(BamHIEcoRISalI)andyouwanttoexpandthatwiththeadditionofNdeI,
    PacI,AscI,andMfeI.Witholigooverlapcloning,youcandesignasetofoligoscontainingyourdesiredrestrictionsitesandaddthemtoyourexisting
    vector.Itisacoupledaysofworkthatwillpayoffforyearstocome.

    Design
    Briefly,wewilldesignoverlappingoligosthatonceannealedcanbecloneddirectlyintotheoverhangsgeneratedbyrestrictiondigestofexistingsites
    intheoriginalvector.

    Designingoligos:
    WewanttoaddNdeI,PacI,AscIandMfeIsitessowewilldesignatopoligowitheachofthesesitesintandem(NdeI
    CATATG
    PacI
    TTAATTAA
    AscI
    GGCGCGCC
    MfeI
    CAATTG
    )andwiththebottomoligobeingthereversecomplimentsothattheyanneal.

    Top:
    5'

    CATATG

    TTAATTAA

    GGCGCGCC

    http://www.addgene.org/plasmidprotocols/annealedoligocloning/

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    Addgene:PlasmidModificationbyAnnealedOligoCloning(withProtocols)

    CAATTG

    3'
    =28bp

    Bottom:
    3'

    GTATAC

    AATTAATT

    CCGCGCGG

    GTTAAC

    5'

    WealsoneedtomakesuretoincludetheadditionalbasesnecessarytocomplementtheoverhangsgeneratedwhendigestingthevectorwithEcoRI
    andSalI(seediagram).Todothisweneedtoadd5'
    AATTC
    and
    G
    3'tothetopoligoand3'
    G
    and
    CAGCT
    5'tothebottomoligomakingourfinaloligos34bpeach.

    Note:Wecouldleaveoffthe3Goneacholigo(andthecomplementaryCoftheotheroligo),butthiswoulddestroytheEcoRIandSalIsitesin
    thefinalvector.
    Top:
    5'

    AATTC

    CATATG

    TTAATTAA

    GGCGCGCC

    CAATTG

    Bottom:
    3'

    http://www.addgene.org/plasmidprotocols/annealedoligocloning/

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    Addgene:PlasmidModificationbyAnnealedOligoCloning(withProtocols)

    GTATAC

    AATTAATT

    CCGCGCGG

    GTTAAC

    CAGCT

    5'

    Orderthefollowingoligosfromyourfavoriteoligosynthesiscompany:
    Top:
    5'AATTCCATATGTTAATTAAGGCGCGCCCAATTGG3'

    Bottom:
    5'TCGACCAATTGGGCGCGCCTTAATTAACATATGG3'

    Note:Ifyouplantophosphatasetreatyourcutvectoritisnecessarytouse5'phosphorylatedoligos.Thisisanoptionthatcanbeaddedwhen
    orderingthemorcanbeperformedenzymaticallylater.

    ExperimentalProcedure
    Digestandpurifyvector:
    1.Whilewaitingforyouroligostoarrive,conductarestrictiondigestof1gofvectorwithEcoRIandSalI
    2.RunanagarosegelandcutoutthebandcontainingyourvectorDNA
    3.GelpurifyyourDNAawayfromtheagaroseusingacommerciallyavailablekitorstandardprotocol.

    Annealoligos:
    Theoligosshouldberesuspendedinannealingbuffer(10mMTris,pH7.58.0,50mMNaCL,1mMEDTA)andmixedinequimolarconcentrations.We
    recommendmixing2geachinatotalvolumeof50Laddadditionalannealingbufferifnecessarytogetto50L.Efficientannealingcanbe
    achievedbyoneoftwomethods:
    Method1.
    Placethemixedoligosina1.5mLmicrofugetube.
    Placetubein9095Chotblockandleavefor35minutes.
    Removethehotblockfromtheheatsource(turnofformoveblocktobenchtop)allowingforslowcoolingtoroomtemperature(~45minutes).
    Method2.
    PlacemixedoligosinaPCRtube.
    Placetubeinathermocyclerprogrammedtostartat95Cfor2minutes.
    Then,graduallycoolto25Cover45minutes.

    Ligation:
    1.Dilute5Lofannealedoligoswith45Lnucleasefreewaterandquantifytheconcentration(shouldbeabout8ng/l).
    2.Mixtheannealedoligoswithcutvectorinmolarratios(vector:insert)between4:3and1:6inastandardligationreaction.(ex.toligatean
    insert5kbinlengthintoa5kbvector,mix100ngofthevectorwith75600ngofannealedoligos)
    3.Transform23Lintoyourfavoritecompetentbacteriaandplate.

    http://www.addgene.org/plasmidprotocols/annealedoligocloning/

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    4.Besuretopickmultiplecoloniesforminipreppingandverifyinsertbysequencing.

    ReferencePage|Top|Index

    http://www.addgene.org/plasmidprotocols/annealedoligocloning/

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