Fast miniprep and denaturation for plasmid
DNA sequencing
[from Y. Yie et al., (1993) NAR 21, 361]
1. Place 1.5 ml of overnight bacterial culture into a 1.5 ml tube
and centrifuge at top speed for 10 sec.
2. Remove the medium by aspiration, leaving the bacterial pellet
as dry as possible.
3. Resuspend the pellet by vortexing in 50 ul of TE buffer (20
mM Tris-HCl, 1mM EDTA). Add 50 ul of phenol:chloroform and vortex
for 10 sec.
4. Centrifuge at top speed for 3 min.
5. Transfer 25 ul of supernatant to a fresh tube, then add 15
ul 2 N NaOH and 110 ul water.
6. Place the tube in boiling water bath for 3 min.
7. Place the tube in a ice-bath, add 20 ul 3 M NaAc (pH5.2) and
2.5 vol. of EtOH, then place the tube in liquid N2 for 5 min.
8. Centrifuge at 12k rpm for 10 min. Rinse the pellet with 70%
ethanol (prechilled) and dry the pellet under vacuum.