DNA priming reaction x ul DNA Enzyme mix 4 ul radioactive nucleotide (12 l) 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].
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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