On-line Methods Ten-minute miniprep

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19 April 2002

 

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

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

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