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 µl 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 µl 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 µl 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 µl), 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 µl 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.
