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.
- Prepare 13 ml tube with 5 ml LB, containing appropriate antibiotic (ampicillin concentration can be up to 400µg/ml).
- Pick up the colony with sterile yellow pipett tip, drop the tip in the tube
- 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.
- Most of the cultures will show sufficient density after 8-10h incubation.
- Transfer 1.5 ml of culture in 1.5 ml tube and centrifuge in table top centrifuge at max speed for 1-2 min.
- Discard the supernatant.
- If the pellet is small repeat 2 previous steps.
- Add 100 µl of P1 buffer (Qiagen) and vortex until the pellet is completely resuspended.
- Add 100 µl of P2 buffer (Qiagen) and mix by shaking in upside-down position.
- Add 100 µl of P3 Buffer (Qiagen) and mix very carefully.
- Centrifuge at 13000 rpm for 15 min. While centrifuging prepare a set of fresh 1.5 ml tubes.
- Transfer the supernatant into a fresh tube, try not to take debris.
- Add 900 µl 100% EtOH, vortex briefly.
- Centrifuge 15 min at 13000 rpm.
- Discard the supernatant and wash DNA Pellet with 500 µl 70% Ethanol.
- Centrifuge 2 min at 13000 rpm.
- Dissolve the DNA pellet in 50 µl of pure H2O.