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        DNA SEQUENCING REACTIONS

        互联网

        1355

        DNA priming reaction

         x ul DNA
          2 ul Reaction buffer, minus DTT
          1 ul primer (20 ng)
          10 ul total
          Heat 90-100℃ 2 min.
          Cool room temp. 30 min.

        Enzyme mix

        4 ul radioactive nucleotide (12 l)
         
        2 ul reaction buffer        ( 6 l)
         
        4 ul water                  (12 l)
         
        4 units enzyme              (1tube)

        Mix DNA and Enzyme mix

        Add 4 ul to 1 ul nucleotides (see nucleotide mixes).

        Incubate 50-60℃ 15 min.

        Add 1 ul chase (0.5mM nucleotides)

        Incubate 15 min.

        Stop and Load

        Reaction buffer (5X) per ml

        0.3 M Tris (pH 8.3) .. 300 ul 1M

        no NaCl ..............

        37.5 mM MgCl2 ........ 37.5 ul 1M

        5 mM DTT ............. 5.0 ul 1M

        water ................ 660 l

        SEQUENASE REACTIONS

        Mix:

        7 ul total of DNA plus water [1~2 g]

        2 ul sequenase buffer

        1 ul primer

        Heat 2 min. at 90~100

        Add 1 ul of 1:4 dilution of s.s. binding protein.

        Leave 30 min. at room temperature.

        While waiting:

        Unfreeze label (35S or 32P dATP)

        Unfreeze Sequenase items

        Labelling mix (one for dGTP; one for dITP if necessary)

        0.1 M DTT

        Stop Mix

        Termination mixes (one set dGTP; one set dITP if necessary)

        Aliquot into tubes marked G,A,T,C:

        2.5 ul of appropriate termination mixes.


        Mix for dGTP reactions:

        11 ul DNA mix

        1 ul 0.1 M DTT

        2 ul labelling mix (1:5 dilution dGTP mix for reading ~500 bp)

        5 ul dATP -32P or -35S

        2 ul 1:8 dilution of Sequenase (dilute with TE)

        Wait 5 min. at room temp.

        Pre-warm termination mixes to 370C

        Add 3.5 ul of labeling mix to each termination mix

        For dITP wait 2-3 min. to add 4 ul stop solution.

        For dGTP around 5 min. is OK.

        This means dITP reactions are best done one at a time.

        dGTP reactions can be done three at a time.

        Add 1l of a 1:10 dilution of 20 mg/ml Proteinase K.

        Heat at 65C for 20 min.

        Before loading onto gel heat around 900C and load immediately.

        To wash short [notched] plate:

        Wash in 2.5% NH4OH, 50% isopropanol, 1/2 cap detergent per 500 ml.

        Then 2X with H2O, and 2X with 95% ethanol.

        Check for good siliconization; if not good, then siliconize with 2% dichlorodimethylsilene in toluene, and repeat wash with water and ethanol.

        To wash long [unnotched] plate:

        Wash in 10 N NaOH. Then 2X with H2O, and 2X with 95% ethanol.

        Acrylamide solution:

        Make 100 ml of 6% (w/v) acrylamide sequencing gel solution with following ingredients:

        48 g urea, 30 ml H2O [to mix], 10 ml 10X TBE, 15 ml acrylamide (38%)/bis-acrylamide (2%), 400 ul 40% (w/v) ammonium persulfate, when completely dissolved bring to final volume with water.

        Filter solution through 0.2 m filter unit. Cool solution on ice for about 15 min [helps to slow polymerization reaction].


        When ready to pour gel, add 60 ul TEMED immediately before casting gel (within 30 sec).

        Let gel polymerize for at least 2-3 hours.

        Pre-electrophoresis:

        Pre-run gel in TBE buffer for 30 min or more at constant power of 45-50 W until temperature reaches 50C.

        Electrophoresis:

        Run gel at 45-50 W at 500C for about 2.5 hr.

        Post-electrophoresis:

        Open plates. Gel should stick to long plate. Fix 35S run in 10% (v/v) acetic acid, 5% (v/v) methanol. Transfer to Whatman paper and dry gel for 30 min at 800C. Expose to X-ray film.

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