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.

9. Remove the cap from the QIAfilter outlet nozzle. Gently insert the plunger into the QIAfilter Midi Cartridge and filter the cell lysate into the previously equilibrated HiSpeed Tip.