DNA priming reaction x ul DNA Enzyme mix Mix DNA and Enzyme mix Add 4 ul to 1 ul nucleotides (see nucleotide mixes). SEQUENASE REACTIONS Mix: While waiting: Termination mixes (one set dGTP; one set dITP if necessary) Mix for dGTP reactions: 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: To wash long [unnotched] plate: 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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