Plasmid Mini-Preps Modified Alkaline Lysis
(Automated Sequence Grade DNA)
Trent Colbert/Hahn lab
last modified 12/12/99
Note: This method works well for DNA sequencing if the vector is a high copy plasmid such as bluescript. However, if the vector is a low copy plasmid such as a pet vector, it is recommended that another method be used to obtain DNA for automated sequencing. For low copy plasmids, we find the Qiagen mini prep kit works well for automated DNA sequencing.
Grow 3 ml of overnight bacterial culture in YT (or LB) plus selective antibiotic. Centrifuge cells in clinical centrifuge in glass tubes (10 min) or in microfuge (2 times 2 min) to harvest the entire 3 ml.
Dump off supernatant from the cells and blot tubes on paper towel to remove remaining liquid. To the pellets add 200 microliers Buffer P1 (the entire cell pellet from the 3 ml culture should be resuspended in a microfuge tube in a total of 200 microliters P1).
Add 200 microliers Buffer P2 to each tube and invert tubes several times but don't vortex.
Add 200 microliters Buffer P3 to each tube and vortex 10 sec.
Spin all tubes 10' in microfuge.
Carefully transfer the supernatant to new tubes.
Add 350 microliters Phenol:CHCl3 (2/1). Vortex the new tubes ~20 sec. and spin 4 min. in microfuge.
Transfer 0.5 ml of the aqueous (top) phase to new tubes being careful not to disturb the interface between the layers. Add 0.7 volume (0.35 ml) isopropanol.
Vortex and freeze on dry ice for 5 min.
Spin tubes 8 min. in microfuge.
Remove supernatant with a drawn out pasteur pipet or pipetman. After removing the supernatant, wait a minute or so and remove any remaining liquid.
Wash pellets with ~300 microliters of 80% ETOH, spin 3 min in microfuge and remove supernatant.
Air dry or speedvac pellets and resuspend in 30 microliters H2O.
For Automated sequencing, use 1-2 microliters (high copy plasmid).
P1: 6.1 g Tris, 3.7 g EDTA-2H2O pH 8.0 w/ HCl/1 liter, add 100 mg/ml RNAse A as needed, usually 10 mg RNase in 100 ml batches, store 4 degrees.
P2: 8.0 g NaOH in 900 ml H2O plus 100 ml of 10 % SDS/1 liter, store R.T.
P3: 294 g KAcetate in 500 ml H2O pH to 5.5 with Acetic Acid (~110 ml), bring to 1 liter, store R.T.
