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Plasmid mini-preparation

Plasmid mini preparation

Plasmid preparation (Quick, Cheap and ABI Sequencing ready)
Minipeps without overnight culture (8 h from colony to plasmid DNA)
Plasmid preparation (Quick, Cheap and ABI Sequencing ready)

Contributed by Dr.K.Schledzewski

Miniprep with Qiagen solutions

  1. Grow up bacterial clone in LB supplemented with appropriate antibiotic for 6-8 h.
  2. Spin 1.5 ml of culture in 1.5 ml tube at 8500 rpm for 1 min discard the supernatant completely and vortex the pellet thoroughly.
  3. Add 125 µl of P1 buffer (Qiagen) and vortex again, check carefully the suspension for any clumbs.
  4. Add 125 µl of P2 buffer (Qiagen) and mix very carefully by inverting the tube no more than 4 times.
  5. Incubate for about 2 min at room temperature (RT).
  6. Add 125 µl of P3 Buffer (Qiagen) and mix very carefully by inverting the tube no more than 4 times.
  7. Incubate for about 5 min at RT, spin down bacterial debris at 15000 rpm for 25 min. While centrifuging prepare a set of fresh microliter tubes.
  8. Carefully transfere 330 µl of supernatant into fresh tube, add 200 mkl of room temperature 2-Propanol, mix by vortexing.
  9. Incubate 2 min at RT, spin down DNA at 15000 rpm for 10 min.
  10. Discard the supernatant and wash DNA Pellet with 70% Ethanol at RT.
  11. Spin down DNA at 15000 rpm for 2 min, remove ethanol completely (an additional centrifugation might be helpful).
  12. Dissolve the DNA pellet in 50 µl of pure H2O.

A standart preparation results in a DNA concentration of 100 to 200 ng/µl.

The usage of the Qiagen Buffer set that costs about 120 Euro and is sufficient for 500-1000 preps is recommended.

Minipeps without overnight culture (8 h from colony to plasmid DNA)

Contributed by Alexei Gratchev

Overday minipreps

  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.
  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.