Plasmid minipreps

Contributed by Dr. Alexei Gratchev

During past 10 years I tested several systems for the isolation of minipreps. This protocol was developed to obtain consitent results using cheap commertially available solutions. Actually I always used rests of solutions from Qiagen plasmid kits. Plasmids isolated with this protocol are pure enough for any further application. We use them for further cloning, amplification and sequencing.

  1. Prepare 13 ml tube with 5 ml LB, containing appropriate antibiotic (ampicillin concentration can be up to 400µg/ml).
  2. Pick up the colony with sterile yellow pipett tip, drop the tip in the tube
  3. Shake your tubers at the rotary shaker as fast as possible (usually 300-400 rpm) at 37° C. Higher temperatures (up to 42° C) can be used for some plasmids and E.coli strains.
  4. Most of the cultures will show sufficient density after 8-10h incubation.
  5. Transfer 1.5 ml of culture in 1.5 ml tube and centrifuge in table top centrifuge at max speed for 1-2 min.
  6. Discard the supernatant.
  7. If the pellet is small repeat 2 previous steps.
  8. Add 100 µl of P1 buffer (Qiagen) and vortex until the pellet is completely resuspended.
  9. Add 100 µl of P2 buffer (Qiagen) and mix by shaking in upside-down position.
  10. Add 100 µl of P3 Buffer (Qiagen) and mix very carefully.
  11. Centrifuge at 13000 rpm for 15 min. While centrifuging prepare a set of fresh 1.5 ml tubes.
  12. Transfer the supernatant into a fresh tube, try not to take debris.
  13. Add 900 µl 100% EtOH, vortex briefly.
  14. Centrifuge 15 min at 13000 rpm.
  15. Discard the supernatant and wash DNA Pellet with 500 µl 70% Ethanol.
  16. Centrifuge 2 min at 13000 rpm.
  17. Dissolve the DNA pellet in 50 µl of pure H2O.

\METHODS\DNA\Plasmid miniprep