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        GELSHIFT

        互联网

        2275

        John Mundy, Institute of Molecular Biology, Copenhagen, Denmark http://www.arabidopsis.org/info/Protocols_Mundy2.jsp#Gelshift 1)Nuclear extract: see nucprep.ptc & nucext.ptc extracts should be at least 3μg/μl

        2)Probe preparation: see endlabl.ptc & isotach.ptc. probe shouldbe 10©20k cpm/μl.

        Fragments larger than 400bp should not be used. Make A stock (25μg/50μl) of probe plasmid digested at one end w/ 5' overhang.

        3)Binding rxn:

        1-2μl probe (0.5ng or 20k cpm in isotach 40mM Tris 7.5 buffer)

        1-2μl poly dIdC or dAdT (3μg/μl in NEB, sonicated to 300©500bp)

        1-6μl extract (5©10μg/μl in NEB)

        >10μl NEB (see nucext.ptc)

        incubate 30' RT

        1μl sequencing dyes immediately prior to loading under tension

        4)Titrations: Start w/ extract titration at 3μg/rxn poly dIdC.

        At extract [] w/ max binding, do dIdC titration.

        work for complete probe binding, none free. Try Mg salts later.

        5)Competitions: Fragments should be isolated by PAGE/isotach. 10-100ng DNA /rxn is usually necessary.

        6)gels:

        Acryl

        48.5ml

        10ml 30/0.8% acryl stock

        1.5ml 10x TBE

        50μl TEMED

        400 μl 10% APS

        run 100©200v w/ circulation

        Acryl/agarose gel

        H2O80ml H2O

        0.7g agarose

        boil to dissolve

        10ml 10x buffer 100mM Tris 7.5, 10mM EDTA, 30mM NaOAc

        10ml 30/0.8% acryl stock

        cool to 60℃

        60μl TEMED

        100μl 10% APS

        let set for 2hr, prerun with circulation 30' at 100v and run w/ circulation

        7)Gels are dried unfixed on Whatman DE 81 sheets at 80℃ on dryer.Expose o/n -70℃ w/screen.

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