Team:Wisconsin-Madison/Plasmid Preparation by Alkaline Lysis

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Plasmid Preparation by Alkaline Lysis Solution 1:
 * 50 mM Glucose
 * 25 mM Tris Cl (pH = 8)
 * 10 mM EDTA (pH = 8)

Solution 2:
 * 0.2 M NaOH (10 M)
 * 1% SDS

Solution 3:
 * 60 mL Potassium Acetate (5 M)
 * 11.5 mL Glacial Acetic Acid
 * 28.5 mL Water

Procedure:

1. Pellet the overnight culture(s) in a 1.5 ml or 2ml eppendorf tube.

2.       Resuspend each pellet in 200 μl Alkaline Lysis Sol I, RnaseA added (final RNase A concentration should be 20 μg/mL)

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

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

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

6.       Rest on ice for 5-10 minutes. 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 chloroform.

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

8.       Pipet 750μl of supernatant/aqueous layer into a fresh tube.

9.       Add 1/10 volume (75μl) 3M NaOAc, pH 5.2. Vortex/flick to mix.

10.   Add 0.7-1.0 Volume COLD isopropanol. Vortex/flick to mix. If in a hurry go straight to step 11, otherwise rest on ice for 10-30 minutes. I have even let it precipitate overnight at 4°C if convenient.

11.   Centrifuge at max. speed for 25 min. Most miniprep protocols say to do this at 4°C, 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.

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

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

16.   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.

17.   Air dry the pellet for ~15 minutes (pellet will change from white to clear as it dries). Resuspend in desired volume of H2O or T10E1, depending on downstream applications

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