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        DNase I Footprinting

        互联网

        8192

         An end labeled DNA probe is incubated with a purified DNA-binding factor or with a protein extract. The unprotected DNA is then digested with DNase I such that on average, every DNA molecule is cut once. Digestion products are then resolved by electrophoresis. Comparison of the DNase I digestion pattern in the presence or absence of protein will allow the identification of a footprint (protected region).

        1. Make the DNA binding reaction by combining the following components in a microcentrifuge tube:

        25 μl of protein extract in HEMG

        10 μl of 10% Polyvinyl Alcohol

        1 μl of 1 M HEPES, pH 7.6

        2 fmol of end-labeled DNA probe (about 1200 cpm)

        1 to 5 μg competitor DNA

        and bring the final reaction volume to 50 μl with ddH2 O.

        2. Mix the components gently and incubate on ice for 10 min (for incubations with purified proteins, incubation temperatures may be increased).

        3. Add 50 μl of Ca/Mg Solution to the binding reaction at room temperature.

        4. Add 1 to 10 μl of DNase I solution (see Hint #1).

        5. Mix quickly and incubate at room temperature for 1 min.

        6. Stop the reaction by adding 100 μl of stop solution and mix by vortexing immediately.

        7. Add 200 μl of phenol/chloroform and mix by inverting several times. Centrifuge at full speed in a microcentrifuge for 10 min to separate the phases. Recover the aqueous phase and transfer it to a fresh microcentrifuge tube.

        8. Add 1 ml of 100% ethanol and mix by inverting several times.

        9. Centrifuge at full speed in a microcentrifuge for 10 min to pellet the DNA and remove the ethanol.

        10. Resuspend the pellet in 70% ethanol and pellet the DNA again. Remove the ethanol and allow the DNA pellet to air dry.

        11. Resuspend the DNA in 6 μl formamide dye and load the sample onto a 6% to 8% sequencing gel (see Protocol on Sequencing Gel Protocol). 

        Competitor DNA    Sonicated Calf Thymus DNA in ddH2 O
        Formamide Dye    0.005% (w/v) Xylene Cyanol FF
        20 mM EDTA
        make up in deionized Formamide
        0.005% (w/v) Bromophenol Blue
        70% (v/v) Ethanol
        1:1 Phenol:Chloroform
        Stop Solution    1% (w/v) SDS
        0.2 M NaCl
        Store at room temperature
        20 mM EDTA, pH 8.0
        0.25 mg/ml carrier RNA
        Ca/Mg Solution    5 mM CaCl2
        10 mM MgCl2
        DNase I    2.5 mg/ml in ddH2 O
        Freeze as 2-5 μl aliquots and store at -70℃
        HEMG    0.1 mM EDTA, pH 8.0
        0.1 M KCl
        25 mM HEPES, pH 7.6 with KOH
        12.5 mM MgCl2
        10% (v/v) Glycerol
        1 mM DTT (add just before use)
        1 M HEPES    pH 7.6 with KOH
        10% (v/v) Polyvinyl Alcohol


        Competitor DNA      Sonicated Calf Thymus DNA in ddH2 O

        Formamide Dye      0.005% (w/v) Xylene Cyanol FF

        20 mM EDTA

        make up in deionized Formamide

        0.005% (w/v) Bromophenol Blue

        70% (v/v) Ethanol

        1:1 Phenol:Chloroform

        Stop Solution      1% (w/v) SDS

        0.2 M NaCl

        Store at room temperature

        20 mM EDTA, pH 8.0

        0.25 mg/ml carrier RNA

        Ca/Mg Solution      5 mM CaCl2

        10 mM MgCl2

        DNase I      2.5 mg/ml in ddH2 O

        Freeze as 2-5 μl aliquots and store at -70℃

        HEMG      0.1 mM EDTA, pH 8.0

        0.1 M KCl

        25 mM HEPES, pH 7.6 with KOH

        12.5 mM MgCl2

        10% (v/v) Glycerol

        1 mM DTT (add just before use)

        1 M HEPES      pH 7.6 with KOH

        10% (v/v) Polyvinyl Alcohol 

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