3.2.2. Polymerase chain reaction
PCR reactions (modified from Dobbelaere et al., 2001) are set up as 50 µl mixtures containing:
- 1-5 µl template DNA
- 50 pmol forward (AFB-F) and reverse primer (AFB-R); (primers used by Govan et al., 1999 also work well)
- 10 nmol of each dNTP
- 1-2.5 U of Taq polymerase in the appropriate PCR buffer containing 2 mM MgCl2.
Use the following PCR conditions: a 95°C (1-15 min) step; 30 cycles of 93°C (1 min), 55°C (30 sec), and 72°C (1 min); and a final cycle of 72°C (5 min).