Dirty Mini Preps

?

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

?

?