DNA PCR reaction

PCR reaction
  1. Dilute the DNA suspension to reach a final concentration of 5 ng/µL (final volume 200 µL); if the samples are thermolysates* centrifuge them at 12 000rpm for 5 minutes to pellet bacterial debris;
  2. Prepare the mix using:
    • 1.5 µL of Taq buffer 10x containing Mg2+
    • 1.5 µL of dNTPs suspension 2mM each
    • 1.5 µL of oligonucleotides suspension 10 mM
    • 3 µL of Betain 5 M
    • 5.5 µL of H2O
  3. Add 2 µL of Taq Polymerase for 8 reactions;
  4. Add 13 µL of the mix to each tube;
  5. Add 2 µL of DNA to each tube;
  6. Centrifuge at 2000 rpm (just reaching this speed and then stopping the centrifuge);
  7. Put the tubes in the thermocycler and chose the program: - EDIT---MAIN---MLVA
    • 94 °C for 5 minutes DENATURATION
    • 94 °C for 30 seconds DENATURATION (1)
    • 60 °C for 30 seconds ANNEALING (2) (1)+(2)+(3) x35 times
    • 72 °C for 45 seconds ELONGATION (3)
    • 72 °C for 10 minutes COMPLETE ELONGATION
    • 20 °C for 5 minutes
  8. Add 1 drop of 10x loading buffer and load 2 µL of each samples on a 2% agarose gel;
  9. Stain the gel with Ethidium Bromide (0.25 microg per ml final concentration; for one liter, 25 µL of a 10mg per ml stock solution
  10. *put 1 loop of bacteria in 200 µL of sterile water; heat at 95 °C for 5 minutes; put on ice for 5 minutes; store at -20 °C until use