Mike's hints for successful minipreping.

1. Always, always, always mark each culture tube in an unambiguous manner (with a sharpie pen).
2. Always, always, always mark each collection and final collection tube in an unambiguous manner (with a sharpie pen). These should correspond to your cultures - all tubes look identical.
3. If you fail to recover plasmid, the most likely cause is that you failed to add ampicillin or that your ampicillin was bad. Repeat using a fresh stock of ampicillin.
4. Always check minipreps by running out 2-4 µL on an agarose gel before setting up restriction digests, etc

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Protocol  Zhou et al 1990 as modified by Tamara Basta

1. Spin 1.5 ml of an overnight bacterial culture for 1 min. (double if the culture is growing slowly)
2. Gently decant supernatant - leave ~50µL of supernatant with the cell pellet
3. Resuspend by running tube along tube rack (Alberto's Trick)
4. Add 300 µL of TENS (lysis buffer) and mix by inversion* until completely lysed -- make up TENS buffer fresh from 10 x stocks (see below).
5. Add 150 µL 3.0 M sodium acetate, pH 5.2 and invert to mix*
6. Centrifuge for 5 minutes pellet cellular debris and chromosomal DNA
7. Transfer supernatant to fresh microfuge tube
8. Add 0.9 mL -20°C 100% ethanol
9. Spin for 5 minutes at 4°C to pellet plasmid DNA
10. Discard supernatant and rinse pellet twice with 70% ethanol
11. Dry pellet in speed-vac for ~5 minutes
12. Resuspend pellet in 50-100 µL in resuspension buffer (you can add RNAase at this step.

* do not vortex at these steps, it will shear the chromosomal DNA

TENS Buffer:  TE + 0.1N NaOH and 0.5% SDS
from 10X TE, 5N NaOH and 20% SDS