DNA Bacterial DNA purification

Bacterial DNA purification
  1. Lyse bacteria (1 colony in 500 µl of TE buffer) in 500 µL of lysis buffer 2X;
  2. Add proteinase K to a final concentration of 100 µg/ml;
  3. Incubate overnight at 37 °C or for 2 hours at 50 °C;
  4. If the suspension is too viscous dilute it in TE buffer (max final volume 400 µL);
  5. Add NaCl to a final concentration of 0,7 M;
  6. Homogenize very well before adding CTAB;
  7. Add CTAB to a final concentration of 0.5 M;
  8. Mix and keep it for 10 minutes at 65 °C;
  9. Extract the precipitated CTAB with 1 volume of chloroform;
  10. Centrifuge at 12 000 rpm for 10 minutes;
  11. Transfer the supernatant in a fresh tube;
  12. OPTIONAL: Purify the DNA using 3 successive extractions with phenol (pH 7.5), phenol-chloroform (1/1), chloroform;
  13. Precipitate the nucleic acid with 2 volumes of ethanol 100%;
  14. Centrifuge at 12 000 rpm for 10 minutes;
  15. Wash with 500 µL of ethanol 70%;
  16. Centrifuge at 12 000 rpm for 5 minutes;
  17. Leave the pellet to dry;
  18. Suspend the pellet in 200 µL of TE buffer;
  19. Determine the DNA concentration using NANODROP.
TE buffer: Pseudomonas aeruginosa lysis buffer: