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CsCl plasmid prep from 500 ml of TB

  1. Grow bacteria O/N in 500 ml of TB.
  2. Spin down cells 10 min. 5000 rpm.
  3. Resuspend cells in 30 ml buffer I + 5mg/ml lysozyme.
  4. Add 60 ml buffer II, mix gently and incubate 5 min at RT.
  5. Add 45 ml buffer III, mix and incubate 10 min on ice.
  6. Spin 15 min at 7000 rpm. at 4°C.
  7. Filter the supernatant through a large kimwipe (fold double twice, then place in a funnel).
  8. Add 60% v/v isopropanol and mix well.
  9. Incubate 10 min at RT.
  10. Spin 15 min 8000 rpm at RT.
  11. Dry the pellet for about 15 minutes at RT.
  12. Resuspend pellet in 8 ml of TE and transfer to a 15 ml Falcon tube.
  13. Weigh the DNA/TE mixture (compare with an empty tube), and add the following amount of CsCl:
    • As many grams as the weight of the DNA/TE mixture.
    • 0.8 gr. for the EtBr in the following step.
    • 0.3 gr. for what you lose in the initial quick spin.
  14. Add 0.8 ml of EtBr (10 mg/ml stock).
  15. Mix well and spin 10 min 3000 rpm.
  16. Transfer supernatant into Beckman Optiseal tubes.
  17. Fill up the tubes with water+CsCl solution (1 gr. CsCl / 1 ml water).
  18. Spin O/N: 55.000 rpm, 17°C in a Beckman Psi60 rotor.
  19. Insert an injection needle in the top of the tube to allow air to enter and take out the DNA band (lower band) with another injection needle (use gauge 20 needles).
  20. Add TE to 5 ml total volume.
  21. Extract the EtBr by mixing the DNA solution with 1 volume water-saturated 1-Butanol and disposing of the upper phase (Butanol + EtBr). Do this a couple of times until the DNA solution is clear.
  22. Transfer the DNA solution into a 15 ml Corex tube (can also do this in Falcon tubes).
  23. Add 2 vol. of 100% EtOH, mix and spin at 10.000 rpm for 15 min.
  24. Wash the pellet with 5 ml. of 70% EtOH and let the pellet dry.
  25. Resuspend the DNA in a suitable volume of TE.

 

Buffer I (store at 4°C)
Glucose 50 mM
Tris-HCl, pH 8.0 25 mM
EDTA 10 mM

 

Buffer II
NaOH 0.2 N
SDS 1%

 

Buffer III
5M Potassium-acetate 60 ml
Glacial acetic acid 11.5 ml
H2O 28.5 ml