Table 3. PCR recipes.
Initial PCRs were undertaken using recipe (a), for optimisation recipes (b)–(d) were used depending on fragment and sample. Reagents that were varied are in bold. Components of column (a) in combination with an annealing temperature of 50 °C worked better for primer pair 4, (d) worked well for primer pair 8, and (c) worked better for some samples in combination with primer pair 2 (Lister et al., 2005). Except for one case, varying the annealing temperature had no influence on the reaction.
| Reagents | Quantity (µl) | |||
|---|---|---|---|---|
| a | b | c | d | |
| PCR Flexi-Buffer (5X) | 2.5 | 2.5 | 2.5 | 5 |
| MgCl2 (25 mM) | 1.5 | 1.5 | 2 | 3 |
| dNTPs (10 mM) | 0.5 | 0.5 | 0.5 | 1 |
| Primer forward (5 µM) | 0.5 | 0.5 | 0.5 | 1 |
| Primer reverse (5 µM) | 0.5 | 0.5 | 0.5 | 1 |
| BSA | 1.3 | 0 | 0 | 0 |
| H2O | 4.6 | 5.9 | 5.4 | 12.9 |
| GoTaq polymerase | 0.1 | 0.1 | 0.1 | 0.1 |
| DNA | 1 | 1 | 1 | 1 |
| Total reaction volume | 12.5 | 12.5 | 12.5 | 25 |