Table 5.
Components and thermocycler program used for the final PCR amplification of the PAM library using Q5 Hot Start High-Fidelity DNA Polymerase. Note the cycle number is reduced from 25 to 8 to reduce errors during amplification.
| Component | Volume (μl) |
Temperature (°C) |
Time (s) |
|---|---|---|---|
| nuclease-free H2O | 28.5 | 98 | 30 |
| 5x Q5 reaction buffer | 10 | 98 | 10 |
| 10 mM dNTPs | 1 | 67 | 20 |
| 10 μM FWD Nextera index (i5) | 2.5 | 72 | 10 |
| 10 μM REV Nextera index (i7) | 2.5 | repeat 8x | |
| purified PCR product | 5 | 72 | 120 |
| Q5 Hot Start High-Fidelity DNA Polymerase | 0.5 | 4–10 | ∞ |