Genomic DNA can be amplified in a 50 µl reaction containing:
- 10 μl of 5x HF buffer (1 5 mM MgCL2)
- 1 U of Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Inc)
- 200 µM of each deoxyribonucleotide triphosphate
- 1 μM forward and reverse primer (Table 2))
- 1 µl of DNA
- initial denaturing for 30 s
- denaturing at 98°C
- 10 touchdown cycles: 98°C for 30 s, 70–60°C (decrease of 1°C per cycle) for 30 s, and 72°C for 30 s,
- 30 cycles of 98°C for 30 s, 60°C for 30 s, and 7 °C for 30 s;
- 10 min extension at 72°C.
Amplified products can be analysed on a 1.5 % (wt/vol) agarose gel stained with ethidium bromide or EZ-vision dye. Alternatively, PCR results could be visualised using DNA imaging. Sharp bands can be sequenced directly (further advice on sequencing and analyses can be found in the BEEBOOK paper on molecular methods (Evans et al., 2013). Note: Alternatively another polymerase can be used and the touch down step can be replaced by adding five normal cycles; however, it will require some optimization.