Alkaline lysis plasmid extraction

Solutions

Alkaline Lysis Solution 1

50 mM glucose

25 mM Tris-Cl (pH 8.0)

10 mM EDTA (pH 8.0)

Prepare Solution I from standard stocks in batches of -100 ml, autoclave for 15 minutes at 15 psi (l.05 kg/cm2 ) on liquid cycle, and store at 4 degrees C.

Alkaline Lysis Solution 2

0.2 N NaOH (freshly diluted from a 10 N stock)

1% (w/v ) SDS

Prepare Solution II fresh and use at room temperature.

Alkaline Lysis Solution 3

5 M potassium acetate - 60.0 ml

Glacial acetic acid - 11.5 mL

H2O - 28.5 ml

The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 4 degrees C and transfer it to an ice bucket just before use.

Protocol

1. Pellet the overnight cultures in a 1.5ml eppendorf tube for 1 min. Spin down 4 to 4.5 ml of culture. (For low copy spin down about 6 ml.

2. Resuspend each pellet in 200ul of Alkaline Lysis Sol I, RnaseA added (final RNaseA concentration should be 100ug/ml) Make sure there are no lumps, homogenized.

3. Add 400ul Alkaline Lysis Sol II. Invert 4-6 times to mix. Do not allow reaction to lyse for more than 5min. Sample should clarify.

4. Add 300ul Alkaline Lysis Sol III. Invert 4-6 times to mix. Sample should have a white precipitate.

5. Add 100ul of chloroform. Do this in a fume hood. Invert 4-6 times to mix.

6. Rest on ice for 5-10min. This step is so that the chloroform does not get too hot in the centrifuges and leak out of the tubes. If you want to skip this step you might consider using less cholorform. I put the tubes at -20 for a couple of minutes.

7. Centrifuge at max. speed (14,000rpm) for 10min.

8. Pipet 750ul of supernatant/aqueous layer into a fresh tube. I do up to 800ul.

9. Add 1/10 volume (75ul) 3M NaOAc, pH 5.2. Flick to mix. 80ul if have 800ul of supernatant.

10. Add 0.7-1.0 volume COLD isopronanol (in freezer). Vortex/Flick to mix. If in a hurry go straight to step 11, otherwise rest on ice for 10-30minutes. I have even let it precipitate overnight at 4C if convenient. 600ul isopropanol. Then I put it at -20C for 5minuetes up to over the weekend if needed.

11. Centrifudge at max. speed for 25min. Most miniprep protocols say to do this at 4C, but I have not noticed decreased yield by centrifuging at room temp.

12. Remove and discard the supernatant. Don’t disturb the pellet. Sometimes I can’t see a pellet and more often than not I still have DNA.

13. Add 1ml of 70% EtOH (at room temp.) Invert 4-6 times to rinse the tube.

14. Centrifuge at max speed for ~5 minutes. Room temp. is fine. Remove and discard the EtOH.

15. Repeat steps 14 and 15 to remove all traces of isopropanol. Pulse spin after removing bulk of final EtOH wash and pipet off remaining EtOH.

16. Air dry the pellet for ~15min (pellet will change from white to clear as it dries). Resuspend desired volume (~30ul) of H2O or EB or T10E1 depending on downstream applications. If you pipet off the EtOH well, then I have done this for as little as 2 min. For fosmids, I usually resuspend the pellet in 20ul water.