You should first run qPCR to adjust template concentration (see real-time PCR).
4X linear PCR:
| Reagent | Initial | Final | 1X reaction (50 μl) | 4.5X (μl) |
| Kapa Buffer A | 10x | 1x | 5 | 22.5 |
| Forward | 10 µM | 0.4 µM | 2 | 9 |
| Reverse (T7) | 10 µM | 0.4 µM | 2 | 9 |
| dNTP mix | 10 mM | 0.4 mM | 2 | 9 |
| Raw library | 2 ng/µL | 0.2 ng/µL | 5 | 22.5 |
| Water | 33.5 | 150.75 | ||
| Kapa Taq | 5 U/µL | 0.5 U/µL | 0.5 | 2.25 |
Nir Lab 