Q5 high-fidelity PCR
High-fidelity PCR using NEB Q5 polymerase. Use whenever you'll clone the product or sequence-validate it. ~280× fewer errors than Taq.
Reaction setup (50 µL)
| Component | Volume | Final |
|---|---|---|
| 5× Q5 reaction buffer | 10 µL | 1× |
| 5× Q5 GC enhancer (optional) | 10 µL | 1× |
| 10 mM dNTPs | 1 µL | 200 µM each |
| 10 µM forward primer | 2.5 µL | 0.5 µM |
| 10 µM reverse primer | 2.5 µL | 0.5 µM |
| Template DNA | variable | 1 pg–10 ng plasmid; 1–250 ng gDNA |
| Q5 polymerase (2 U/µL) | 0.5 µL | 0.02 U/µL |
| Nuclease-free H₂O | to 50 µL | — |
Thermocycler program
| Step | Temp | Time | Cycles |
|---|---|---|---|
| Initial denaturation | 98 °C | 30 s | 1 |
| Denaturation | 98 °C | 10 s | 25–35 |
| Annealing | Ta (see below) | 20–30 s | |
| Extension | 72 °C | 20–30 s/kb | |
| Final extension | 72 °C | 2 min | 1 |
| Hold | 4–10 °C | ∞ | — |
Annealing temperature
Use the NEB Tm calculator with "Q5" selected. As a rule, Ta = Tm of the lower-Tm primer (NEB calculator already accounts for Q5's higher salt). For overhangs longer than ~20 nt, use the Tm of the matched (annealing) portion only for the first 5 cycles, then anneal at Tm of the full primer for the rest.
Notes
- Q5 is 5′→3′ exonuclease-deficient and produces blunt ends. Don't use TA cloning directly with Q5 product — clean up and add an A-tail with Taq if needed.
- For GC-rich templates (≥ 65% GC), include the GC enhancer.
- For very long products (> 6 kb), use Q5 Hot Start and increase extension to 50 s/kb.
- Always include a no-template control (NTC) — use water in place of template.
- If you see smearing, drop primer concentration to 0.25 µM or shorten extension.