DNA digestion

DNA digestion with restriction enzymes (for examples Hind III and Eco RI)
  1. Choose the buffer for the enzymes:
    • buffer B for HindIII
    • buffer H for EcoRI
  2. Take out from the fridge the Eppendorf containing the purified phage DNA;
  3. Mix in a single tube the following solutions for each digestion reaction:
    • 15 µL of H2O
    • 2 µL of buffer
    • 3 µL of enzyme
  4. Mix and transfer 20 µL of the mix in each Eppendorf labeled with the name of the phage and the enzyme used for the digestion;
  5. Add 5 µL of phage DNA to each eppendorf;
  6. Incubate at 37 °C for 12-16 hours;
  7. Prepare a 0.8 % agarose gel;
  8. Add 10x loading buffer (Blue) to each Eppendorf tube and incubate at 65 °C for 10 minutes;
  9. Put tubes on ice until loading the digested samples on the gel;
  10. Stain the gel in a solution containing TBE buffer 0.5X and 3 drops of EtBr (0.25 µg per ml final concentration);
  11. Load 1.5 µL of lambda phage genome digested with HindIII restriction enzyme plus 1 drop of Blue or 2 µL of 1 kb-ladder as molecular weight.