Miniprep - Kit-free high throughput protocol

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Revision as of 00:06, 23 September 2009 by Vaishnavi Ananth (Talk | contribs)
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Solutions/reagents:

Equipment:

  • Centrifuge
  • Flasks of appropriate volumes
  • Eppendorf tubes

Steps:

  1. Measure out 1.5 ml of overnight culture containing your plasmid into an Eppendorf tube.
    Centrifuge at a speed of 5000 rpm for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
  2. Add 300 µl of STET buffer.
    Resuspend the pellet by vortexing/by shaking vigorously.
  3. Add 10 µl of lysozyme (10mg/ml).
    Vortex the mixture for a few secs.
    Immerse in boiling water for 40 secs.
  4. Centrifuge at maximum speed for 30 mins at 4°C, gently aspirate out the supernatant and discard it.
  5. Remove pellet from each tube with a toothpick. The cellular debris should stick well to the toothpick. Try to insert and remove the toothpick from the center of the tube so you don't get any cellular debris on the sides of the tube.
  6. Add 300 µl of ice-cold isopropanol.
    This is done to precipitate the DNA.
  7. Centrifuge at maximum speed for 10 mins at 4°C, gently aspirate out the supernatant and discard it.
  8. Add 200 µl of ice-cold 80% ethanol.
    This is to wash the pellet.
    Centrifuge at maximum speed for 5 mins at 4°C, gently aspirate out the supernatant and discard it.
  9. Option 1: Dry the pellet in air.
    (or)
    Option 2: Dry the pellet in a speedvac.

  10. Add 50 µl of TE buffer.
    Resuspend the pellet by vortexing/by shaking vigorously.

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