碱裂解法分离质粒DNA

Reagents:

Soln 1: 50 mM glucose/10 mM EDTA/25 mM Tris pH 8. Autoclave before use. Add 2 mg/ml Lysozyme just prior to use.

Soln 2: 0.2 N NaOH/1% SDS. Keep solution at room temperature. Solution is normally prepared from 10X concentrated solutions the day of use.

Soln. 3 (A): 3 M Acetate (room temperature) Dissolve 3 M sodium acetate in minimal water. Adjust pH to 4.8 with glacial acetic acid. Adjust volume to 1 liter. To make 500 ml: use about 100 ml water, adjust pH to 4.8 , then adjust volume to 500ml with water.

Soln 3 (B): per 100 mls:29.4 g potassium acetate/5 ml 90% formic acid/add water to bring to final volume. Filter if turbid.

Protocol:

Culture Volume30-35 ml1.5 ml500 ml 1. Centrifuge to pellet and add soln 1 on ICE for 15-30 min. 3.5 ml100 µl20 ml 2. Add soln. 2, mix gently on ice for 5 min. 7 ml200 µl40 ml 3. Add soln 3, mix by inversion and leave on ice for 30-60 min. 5.25 ml150 µl30 ml 4. Centrifuge for 15 min at 12,000 x g 5. Take supernatant and add 2 vol. cold (-20°C) ethanol. Maintain at -70°C for 5 min. 6. Centrifuge for 5 min at 12,000 x g 7. Resuspend pellet in 0.1 M sodium acetate- 0.05 M Tris, pH 8.0. Precipitate with 2 volumes of cold ethanol as before. 1-2 ml100 µl10 ml 8. Resuspend pellet in TE buffer and then 1/3 volume 7.5 M ammonium acetate. Precipitate DNA with 2 volumes of cold ethanol. Dry pellet under vacuum. Step number 7 can be omitted if needed. Pellet can also be washed 70% ethanol to remove salt prior to drying.

Modification of Birnboim and Doly

Additional solution: 5 M Lithium chloride, 0.05 M MOPS, pH 8.0. Filter sterilize and protect from light with aluminum foil. Can be stored at room temperature.

1. Dissolve pellet from step 6 above in TE buffer and add an equal volume of the litium chloride solution.

2. Maintain at 65°C for 15 min. and then centrifuge for 15 min at 12,000 x g.

3. Save supeRNAtant and precipitate by adding 2 vol. of cold ethanol, etc.

4. Collect pellet and reprecipitate in TE-ammonium acetate as above.