Alkaline Lysis Plasmid Prep（碱裂解法制备质粒DNA）

Alkaline lysis miniprep

1. Grow bacteria overnight in 37° shaking incubator, with lids very loose and taped on. I normally use 5 ml of liquid medium in a 50ml conical bottom tube. Be sure to include the appropriate selective antibiotic.

2. Remove 1 ml of culture from each tube and mix it with 1 ml of freezedown solution (65% glycerol, 0.1 M MgSO4, 25mM Tris pH 8, autoclaved) in a cryo tube. Freeze at

-85°C. (This is optional if you already have a freezedown of the clone).

3. Centrifuge the remainder of each culture at 1500xg for 5 min. Pour off the supernatant and resuspend the bacterial cell pellet in 200 %26micro;l of GTE buffer (50 mM glucose, 25 mM Tris pH 8, 10 mM EDTA). If you don’t want RNA, add 1 ul of 1 mg/ml RNAse to each suspension. Transfer the resuspended cells to a microcentrifuge tube.

4. After 5 min at room temperature, add 400 %26micro;l of freshly prepared alkaline solution (0.2 N NaOH, 1% SDS) and mix by inverting several times. (to prepare alkaline solution, mix 2 ml 1 N NaOH, 1 ml 10% SDS, and 7 ml H2O).

5. After 5 min on ice, add 300 %26micro;l of 7.5 M ammonium acetate solution. Mix gently for a few seconds. Keep tube on ice for 10 min.

6. centrifuge at 10,000 RPM for 3 minutes.

7. transfer supernatant to a clean microcentrifuge tube, discard pellet.

8. repeat steps 6 and 7.

9. Add 0.6 volumes of isopropanol (400 to 500 %26micro;l), mix, and incubate 10 min at room temperature.

10. centrifuge at 21,000 RPM in high-speed centrifuge for 15 min.

11. Discard supernatant and add 1 ml of ice-cold 70% ethanol. Do not disturb the pellet; centrifuge at 21,000 RPM for 10 min.

12. Discard supernatant and speed-vac about 3 min, or until no alcohol remains.

13. Dissolve pellet in 20 %26micro;l of TE (10 mM Tris pH 8, 1 mM EDTA).

14. Quantify spectrophotometrically.

Mostly stolen (with minor modifications) from

Morelle, G. 1989. A plasmid extraction procedure on a miniprep scale. Focus 11(1): 7-8.