Qiagen MIDI protocol

1. Pick a single colony from a streak plate and inoculate 25 ml of LB with appropriate antibiotic. Incubate overnight.

2. Get an ice bucket ready and put buffer P3 (stored in 4°) in it. Pour the culture into a 50 ml conical polypropylene tube and centrifuge for 10 min at 2000 x g in the C0650 rotor on the Beckman. (or you could split it into a couple of 15 ml conicals and spin at top speed in the clinical centrifuge for 15 min).

3. Discard the supernatant. Resuspend the cells in 4 ml of buffer P1 (stored in 4°) by vortexing repeatedly, leaving no clumps. Transfer the suspension into a round-bottom 13 ml polycarbonate tube.

4. Add 4 ml of buffer P2 (stored in Qiagen buffers box) and mix by covering with parafilm and inverting 4-6 times. Incubate at room temp for 5 min.

5. Add 4 ml of cold buffer P3. Cover with parafilm immediately and mix by gentle inversion 4-6 times. Incubate on ice 15 min.

6. Centrifuge in the F0650 rotor, using a blue rubber adapter sleeve, at 20,000 x g for 30 min in the Beckman. Promptly pour supernatant into another round-bottom 13 ml polycarbonate tube and re-centrifuge at 20,000 x g for 15 min. While the centrifugation is happening, equilibrate a Qiagen-tip 100 (see next step).

7. To equilibrate the tip, put it in a plastic collar and put this assembly into the top of a 50-ml conical polypropylene tube. Add 4 ml of buffer QBT and let it drip through (takes about 5 min).

8. Pour the supernatant from step 6 into the equilibrated column and let it drip through. The plasmid DNA will adsorb onto the column.

9. Wash the column by adding 10 ml of QC buffer and letting it drip through. Repeat with another 10 ml. You may need to discard the washes out of the 50 ml tube sometime during this.

10. Move the tip and collar assembly to a new 50-ml tube. Add 5 ml of buffer QF and let it drip through into the new tube. This eluate contains the plasmid DNA.

11. Pipette 1-ml aliquots of the eluate into microfuge tubes. To each one, add 700 ul of room-temperature isopropanol. Mix and immediately centrifuge in the F2402 rotor at 21,000 x g for 15 min.

12. Pour off supernatant. Add 1 ml cold 70% ethanol to each tube and spin at 21,000 x g for 10 min in the F2402 rotor. Don’t disturb the pellet.

13. Pour off ethanol and dry the tubes in the speed-vac for 2-4 min, or until no ethanol smell is detectable. Be sure not to let the pellet dry entirely.

14. Add 20 ul of diH20 or TE to each tube to redissolve DNA. TE is safer for long-term storage, but may inhibit some enzymatic reactions.