This is a cached page for the URL ( To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
"Dirty" Mini Preps:

"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 l Buffer P1 w. RNase A


4)       Add 300 l 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 l 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 l 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 l aqueous phase to new tube.


9)       Add 0.7 volumes (560 l) 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 l Te buffer.


12)    Test 2 l by restriction digestion.



Buffer P1 (store at 4C):

50 mM Tris-HCl pH 8.0

10 mM EDTA

100 g/ml RNase A


Buffer P2:

200 mM NaOH

1% SDS


Buffer P3:

3.0 M potassium acetate pH 5.5



JLA, 8/3/07