DNA Phage DNA preparation

Phage DNA preparation
  1. Prepare the bottom agar using 4 g of agarose and 250 mL of LB medium;
  2. Mix 100 µL of bacterial suspension with 10 µL of phage suspension (MOI 1:1000) and wait for 15 minutes at room temperature;
  3. Pour the mix on the plate using 4 mL of top agar;
  4. Incubate the plate for 8 hours at 37 °C;
  5. Add 5 mL of SM phage buffer to the plate and leave overnight at 4 °C;
  6. Take 4 mL from each plate and add pancreatic RNase (final concentration 10 µg/mL) and wait for 15 minutes at room temperature;
  7. Add:
    • 0.4 mL of EDTA 0.5 M
    • 0.2 mL of SDS 10%
    • 0.2 mL of Tris 2 M
    • 10 µL of diethylpyrocarbonate (DEPC)
  8. Remove the cap and incubate in a pre-warmed-waterbath at 65 °C for 30 minutes;
  9. Keep the tube on ice for 1 hour and add 1 mL of potassium acetate 5 M;
  10. Using the sigma rotor ref. 12139 centrifuge at 14 000 rpm for 20 minutes at 4 °C;
  11. Transfer the surnatant in a new tube;
  12. Add 2 volumes of ethanol 100 %;
  13. Keep the tube at -20 °C for 1 hour;
  14. Centrifuge at 14 000 rpm for 20 minutes at 4 °C;
  15. Discard the surnatant;
  16. Add 4 mL of ethanol 70 % and centrifuge at 14 000 rpm for 5 minutes at 4 °C (do it twice);
  17. Leave it to dry for 24 hours without the cap;
  18. Add 400 µL of TE (Tris-EDTA) buffer and wait for 15 minutes;
  19. Transfer the suspension in a new Eppendorf tube and measure DNA concentration using NANODROP;
  20. Store at -20 °C until use.