Miniprep - Kit-free high throughput protocol

From OpenWetWare

Revision as of 01:50, 20 November 2009 by Vaishnavi Ananth (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Solutions/reagents:

Equipment:

  • Centrifuge
  • Eppendorf tubes

Steps:

  1. Measure out 1.5 ml of overnight culture containing your plasmid into Eppendorf tube (1).
    Centrifuge at a speed of 5000 rpm for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
  2. Measure out 300 µl of STET buffer into Eppendorf tube (1).
    Resuspend pellet by vortexing/by shaking vigorously.
  3. Add 10 µl of lysozyme (10mg/ml).
    Vortex the mixture for a few secs.
    Store at 100°C 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 pellet by vortexing/by shaking vigorously.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 50 mins

Personal tools