Team:Lyon-INSA-ENS/Realisation/Protocols/DNA-purification

Plasmid or Cosmid DNA Purification Using HiSpeed Plasmid Midi Kits

This protocol is for preparation of up to 200µg of high- or low-copy plasmid or cosmid DNA using the HiSpeed Plasmid Midi Kit.

A final DNA concentration of up to 0.4µg/µL can be expected, if eluting a high-copy plasmid with 500µL of Buffer TE.

Procedure

1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2-5mL LB meduim containing the appropriate selective antibiotic. Incubate for approx. 8h at 37°C with vigorious shaking (approx. 300 rpm).

2. Dilute the starter 1/500 to 1/1000 into selective LB medium. For high-copy plasmids inoculate 50mL medium. Grow at 37°C for 12-16h with vigorous shaking (approx. 300 rpm).

3. Harvest the bacterial cells by centrifugation at 6000 xg for 15 min at 4°C.

4. Resuspend the bacterial pellet in 6 mL Buffer P1.

5. Add 6 mL Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4-6 times, and incubate at room temperature (15-25°C) for 5 min. During the incubation prepare the QIAfilter Cartridge. Screw the cap onto the outlet nozzle of the QIAfilter Midi Cartridge. Place the filter into a convenient tube or a QIArack.

6. Add 6mL chilled Buffer P3 to the lysate, and mix immediately and thoroughly by vigorously inverting 4-6 times. Do not incubate the lysate on ice.

7. Pour the lysate into the barrel of the QIAfilter Cartridge. Incubate at room temperature for 10 min. Do not insert the plunger !

8. Equilibrate a HiSpeed Midi Tip by applying 4 mL Buffer QBT and allow the column to empty by gravity flow.