10 Min Miniprep

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Harvesting:

1. Transfer a single bacterial colony into 5 ml LB broth containing the appropriate antibiotic. Incubate the culture overnight at 37˚C with vigorous shaking.

2. Pour 1.5 ml of the culture into a microfuge tube. Store the remainder of the culture at 4˚C.

10 Min Miniprep:

1. Centrifuge 1.5 ml of the culture for 10 sec at room temp in a microfuge.

2. Decant supernatant gently, leaving 50-100 µl together with pellet, and vortex for 5 sec to resuspend the pellet completely.

3. Add 300 µl TENS (TE containing 0.1M NaOH and 0.5% SDS) and vortex for 2-5 sec.

4. Add 150 µl of 3.0 M NaOAc (pH 5.2) and vortex 2-5 sec.

5. Spin 2 min to pellet cell debris and chromosomal DNA.

6. Transfer to a new tube.

7. Add 2 volumes (0.9 ml) of cold 100% EtOH (pre-cooled to –20˚C) and mix by vortexing for 2-5 sec.

8. Centrifuge for 2-3 min at 4˚C to pellet plasmid DNA and RNA.

9. Remove the supernatant, add 1 ml cold 70% EtOH, spin for 3 min at 4˚C, and decant the supernatant.

10. Dry the pellet.

11. Re-dissolve in 30 µl sterile 1X TE (pH 8.0) or H2O. Vortex briefly and store at –20˚C.