I received a ton of mail, so I will post my version of the protocol
here. Please note it is very similar to the protocol published by C.
Xiang, H. Wang, P. Shiel, P. Berger and D.J. Guerra. I had developed
this protocol about 1 year ago for my convenience. Turns out it is very
similar to their method. At any rate, the protocol is....
1. Grow cells (I used DH5-alpha) in 4 mls LB Broth with appropriate
selective antibiotics overnight (37 C, with shaking).
2. Fill Eppendorf tube completely and spin in microfuge about 20 seconds.
3. Pour off supernatant and resuspend in 100 microL of GTE (50 mM
glucose; 25 mM tris-HCl, pH 8.0; 10 mM EDTA, pH 8.0) with vortex.
4. Add 200 microL lysis solution (0.2 M NaOH, 1% SDS) and invert tubes a
5. Add 150 microL of 5M potassium acetate and mix by rapid shaking. Make
the acetate solution by mixing 60 ml 5M potassium acetate, 11.5 ml
glacial acetic acid and 28.5 ml water. NOTE: This is their recipe, which
works a little better than what I have used.
6. Centrifuge 1 min at high speed.
7. With a sterile toothpick, remove the pellet (it should be relatively
solid and stable for easy removal).
8. Add 1 ml 95% EtOH and invert to mix (I have successfully used both 95%
9. Centrifuge 1 min to pellet, and dry pellet.
10. Resuspend in TE (I have used up to 150 microL to resuspend and still
obtained a high concentration of plasmid DNA) with 25 microgram/ml
RNase. I have a stock of TE with this concentration of RNase already
added that seems to work well.
I have used this prep as is for sequencing and restriction digests and
cloning. Good luck.