The standard method is to use
GFX plasmid purification column from Amersham/Pharmacia. But for a more
traditional method see below:
Grow plasmid from bug stocks or from plate colony with the appropriate
antibiotic in 4ml of LB overnight in loose capped 15 ml tube, shaking at
37°C. This will give a yield of 50-150µg plasmid.
Spin for 5min at 5000g.
Resuspend the pellets by adding 190µl of water and transfer 200µl
to a microtube.
Add 400µl of freshly prepared 1% SDS/0.1M NaOH, and gently mix by
inversion several times. Cells should lyse and form a homogenous, viscous
mixture. Add 300µl of 3M potassium acetate/2M acetic acid, 400µl
phenol, pH 8.0, 200µl chloroform and vortex thoroughly.
Spin in a microfuge at full speed for 5min and transfer 750µl of
supernatant to a fresh tube.
Precipitate by adding 700µl of isopropanol. Vortex vigorously and
leave at -20°C for 20min.
Spin in a microfuge at full speed for 10mins and, after removing all traces
of isopropanol, resuspend the white pellet in 100µl 20µg/ml
RNAaseA. Leave for 10mins at room temperature. Add 100µl of 13% PEG/1M
NaCl and precipitate on ice for 20min. The PEG preciptation stage removes
all low Mwt material.
Spin at full speed for 10min, wash with 200µl of 70% ethanol by pippetting
it into the tube, leaving it for a minute and carefully pippetting it all
out again, being careful not to disturb the pellet, which may not be visible
at this stage. Dry on the bench with lid open for 5min and resuspend in
50µl of water.
Notes- 0.5µl of a plasmid prep is usually more than sufficient
for an agarose gel loading. This method may be scaled up or down as required.
2µl of these plasmid preps may be used directly for ABI sequencing.