Polymerase Chain Reaction (PCR)

Components:

- Millipore H2O (sterile);
- 20 x dNTP solution (in the stock solution the concentration of each nucleotide is 5 mM);
- downstream primer (stock solution in H₂O: 10^-5 M);
- upstream primer (stock solution in H₂O: 10^-5 M);
- template DNA (1 mg - 100 ng);
- Taq polymerase;
- 10 x PCR buffer (sold with Taq polymerase);

- Final volume should be 50 µl.
- Mix the components reported above using the following amounts:

- If using degenerated primers use 4 µl instead of 2 !

- If 0.5 ml tubes is used, PCR solution is overlaid with 2 drops of mineral oil .

-Reactions are performed on a thermal cycler with the following temperature conditions:
94 °C (3 min) - [94 °C (1 min) - T_M (1 min) - 72 °C (1 min)]25 - 65 °C (2 min)
- T_M is the annealing temperature and it is calculated with the formula:
T_M + 69.3 + [41 x (number of G and C content) - 650]/ primer lenght.
- Usually T_M is comprised between 55 °C and 62 °C.

-The elongation time (72 °C) depends on the the fragmentís lenght that is going to be amplified. It is 45 seconds for 1 kb fragment, 1 minute for 1.5 kb and 2 minutes for 3 kb.

IMPORTANT: as negative control no DNA template is introduced into the PCR mixture