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        DNA SEQUENCING REACTIONS

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        1045

        DNA priming reaction

        x ul DNA
              2 ul Reaction buffer, minus DTT
              1 ul primer (20 ng)
              10 ul total
              Heat 90-100C 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 50-60C 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 90-100C
              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 37C

        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 90C 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 50C 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 80C. Expose to X-ray film.  

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