1. Place 1.5 ml of LB media (with appropriate antibiotic if needed; for ampicillin resistant bugs, use 40-100 µg/ml ampicillin) in a sterile tube such as 14 ml snap-cap tubes. Incoulate from a single bacterial colony, and shake overnight at 37˚.
2. The next day, decant the culture into 1.5 ml microfuge tubes. Spin down in microcentrifuge, at max. speed (~12,000 rpm), 5 min.
3. Decant or aspitrate the cleared media from bacterial pellet . Invert the tube and tap briefly on paper towels to “drip-dry”.
4. Add 200 µl cold (4˚) Solution I. Vortex to resuspend cells completely; make sure no clumps of cells remain.
5. Add 200 µl fresh Solution II. Do not vortex. Invert tubes about 6 times to lyse the cells. The lysate should become relatively clear.
6. Add 200 µl cold (4˚) Solution III. Do not vortex. Invert tubes about 6 times to thoroughly mix. A dense fluffy white precipitate should form.
7. Spin down in microcentrifuge, at max. speed (~12,000 rpm), 5 min.
8. Transfer 500 µl of the supernatant to a fresh tube, carefully avoiding solid clumps. Discard the tube with the dregs of liquid and pelleted material.
9. Add 1.0 ml of cold (-20˚C) 100% ethanol. Mix well by inverting and brief vortexing. (Optional: incubate at –20˚ for 30 minutes which can increase final yield). Spin down in microcentrifuge, at max. speed (~12,000 rpm), 15 min.
10. A pellet should be readily visible. Decant supernatant. Invert the tube and tap briefly on paper towels. Add 100 µl of cold (-20˚) 70% ethanol. Spin down in microcentrifuge, at max. speed (~12,000 rpm), 2 min.
11. Remove all of 70% ethanol supernanant carefully from the pellet. Leave tubes open on benchtop for ~5 min to dry briefly. Add 25 µl TE pH 7.5. Pellets should readily dissolve.
For plasmids, 1 µl is usually plenty for a restriction digest. For BACs, use at least 5-10 µl per digest. Since BACs are low copy vectors you may need to prep more than 1.5 ml of culture to get enough DNA. Simply scale up the protocol accordingly.
Solutions for alkaline lysis:
Solution I (a.k.a. GTE) + RNAse A 100 ml 500 ml
50 mM Glucose 0.9 g 4.5 g
25 mM Tris pH 8.0 2.5 ml 1 M 12.5 ml 1 M
10 mM EDTA 2.0 ml 0.5 M 10.0 ml 0.5 M
RNAse A 50 µg/ml 0.5 ml of 10mg/ml 2.5 ml of 10mg/ml
ddH20: to 100 ml to 500 ml
Filter-sterilize and store at 4˚C.
Solution II 100 ml - Make fresh each time.
0.2 M NaOH 2 ml of 10 M stock
1% SDS 10 ml of 10% stock
ddH20: to 100 ml
For 1 liter dissolve 294.5 g potassium acetate in approx. 500 ml ddH20. Add glacial acetic acid to lower pH to 5.5 (will require 150-200 mls). Bring final volume up to 1 liter with ddH20. Store at 4˚ C.
TE pH 7.5:
10 mM Tris, diluted from 1M pH 7.5 stock
1 mM EDTA, diluted from 0.5M pH 8.0 stock