1)       Grow 3-5 ml over-night E. coli cultures containing your plasmid

2)       Spin down 1.5 ml cells

3)       Resuspend in 300 µl Buffer P1 w. RNase A

4)       Add 300 µl Buffer P2.

Mix by inverting tube a couple of times (no vortex). It is important that cells are completely lysed. Incubate at RT, 5 min.

5)       Add 300 µl ice cold Buffer P3, mix by inverting a couple of times (no vortex). Incubate on ice, 5 min or more.

6)       Spin 14,000 rpm, 10 min.

7)       Transfer supernatant to new tube. Extract with 500 µl PCA (Phenol:Chloroform:Iso-amyl alcohol, 50:49:1). Spin 14,000 rpm, 5 min.

Note: The PCA step is optional if the preps are used for restriction digests - however, the plasmid DNA is not likely to be stable over time without PCA extraction due to potential co-purifying DNases.

8)       Transfer 800 µl aqueous phase to new tube.

9)       Add 0.7 volumes (560 µl) isopropanol. Mix by inversion. Spin 14,000 rpm, 15 min at 4˚C.

10)    Remove isopropanol. Wash with 70% EtOH (careful, pellets sometimes come loose). Spin 14,000 rpm, 2-5 min at 4˚C. Remove all EtOH.

11)    Air dry. Resuspend in 20 µl Te buffer.

12)    Test 2 µl by restriction digestion.

Buffer P1 (store at 4C):

50 mM Tris-HCl pH 8.0

10 mM EDTA

100 µg/ml RNase A

Buffer P2:

200 mM NaOH

1% SDS

Buffer P3:

3.0 M potassium acetate pH 5.5