Cosmid DNA

CsCl Cosmid DNA Prep

Based on a protocol I got from Steve Myster, who got it from Mingang Li. I modified it a bit to suit my own preferences.

  1. Grow a 500 ml overnight culture with appropriate antibiotic.
  2. Pellet cells in 1L bottles in the IEC at 2.5K rpm for 15 minutes.
  3. Pour off the supernatant. Drain the pellet well by inverting the bottle on a paper towel for a short time.
  4. Resuspend the cells in 20 ml TEG. Transfer to a 250 ml round bottom centrifuge bottle. Incubate 5-10 min at room temperature.
    [Stock] Reagent [Final] Amount
    2 M Glucose 50 mM 2.5 ml
    1 M Tris-HCl (pH = 8) 25 mM 2.5 ml
    0.5 M EDTA (pH = 8) 10 mM 2 ml
    — H20 — 93 ml
    Total 100 ml
  5. Add 40 ml of freshly prepared alkaline lysis solution. Mix thoroughly but gently by swirling or inverting several times. Incubate 10 min on ice.
    [Stock] Reagent [Final] Amount
    10 M NaOH 0.2 M 2 ml
    20% SDS 1% 5 ml
    —
    H2O —
    93 ml
    Total 100 ml
  6. Add 30 ml ice-cold 7.5 M ammonium acetate solution. Incubate on ice for at least 10 min.
  7. Centrifuge in the IEC at 2.5K rpm for 15 minutes.
  8. Transfer the supernatant into clean 250 ml round bottom centrifuge bottle. The pellet is not hard, so take care when pouring but don’t worry if some debris is transferred with the supernatant. Note: Increasing the spin time doesn’t appear to make the pellet more cohesive.
  9. Add 55 ml isopropanol, mix well and let stand 15 min at room temperature.
  10. Spin in the IEC at 2.5K rpm for 15 min.
  11. Rinse pellet with 100 ml 70% ethanol.
  12. Invert bottle and allow DNA pellet to dry for at least 15 minutes.
  13. Redissolve pellet in 3.5 ml TE. Try to avoid creating bubbles as this will make accurate volume measurement difficult. Transfer to a graduated 15 ml conical tube and bring volume to exactly 4.0 ml with TE.
  14. Add 4.27 g CsCl, Shake gently to dissolve and try to avoid introducing bubbles.
  15. Add 120 µl of 10 mg/ml Ethidium Bromide ([Final] = 300 µg/ml) to 1/2″ x 2″ quick-seal tubes.
  16. Transfer the DNA solution into the quick-seal tube using a Pasteur pipet.
  17. Spin in a VTi 65 rotor in a Beckman L5 centrifuge at 22°, overnight at 45K rpm or 4 hours at 60K rpm. It is OK to use the brake when stopping the run.
  18. Visualise the DNA bands using an UV light. Use an 18 ga. needle and 1 ml syringe to pull the lower (supercoiled) DNA band. The expected volume of material pulled from the quick-seal tube is 500 to 700 µl.
  19. Extract the ethidium by adding an equal volume of NaCl-saturated isopropanol. Mix gently by inverting several times. Spin briefly in a microcentrifuge and remove the pink upper band. Repeat until the upper layer is clear.
  20. Separate the sample into 200 µl (X µl) or smaller aliquots. Add 3X µl of water and mix gently. Add 0.06 new volumes (0.06 x 4X) of 7.5M ammonium acetate and 0.7 new volumes (0.7 x 4X) of isopropanol. Mix gently.
  21. Allow the DNA to precipitate at 4° overnight.
  22. Spin at top speed in a microcentrifuge for 15 minutes at 4°.
  23. Drain off the supernatatant and wash the pellet with 70% EtOH.
  24. Dry the pellet and resuspend in TE.
  25. Quantitate the [DNA] by taking a OD260 reading of a diluted sample of the DNA.

[DNA] = OD260 x dilution x 100 x 0.001 = µg/µl

This page is, as always, Copyright (c) 1997 by Craig D. Amundsen. All rights reserved.