PCR Using Q5® Hot Start High-Fidelity DNA Polymerase (M0493)
New England Biolabs
Abstract
Q5®Hot Start High-Fidelity DNA Polymerase is a high-fidelity, thermostable, hot start DNA polymerase with 3´→ 5´ exonuclease activity, fused to a processivity-enhancing Sso7d domain to support robust DNA amplification. The addition of an aptamer-based inhibitor allows room temperature reaction setup. With an error rate ~280-fold lower than that of Taq DNA Polymerase, Q5 Hot Start High-Fidelity DNA Polymerase is ideal for cloning and can be used for long or difficult amplicons. Q5 Hot Start High-Fidelity DNA Polymerase is supplied with an optimized buffer system that allows robust amplification regardless of GC content. The 5X Q5 Reaction Buffer contains 2 mM Mg++at final (1X) reaction concentrations and is recommended for most routine applications. For GC-rich targets (≥ 65% GC), amplification can be improved by the addition of the 5X Q5 High GC Enhancer. Q5 Hot Start High-Fidelity DNA Polymerase is unlike typical, lower fidelity PCR enzymes. To determine the optimal annealing temperatures for a given set of primers, use of the NEB TmCalculatormCalculatoris highly recommended.
Before start
Please note that protocols with Q5® Hot Start High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.
Steps
Set up the following reaction:
| A | B | C | D |
|---|---|---|---|
| Component | 25 µl Reaction | 50 µl Reaction | Final Concentration |
| 5X Q5 Reaction Buffer | 5 µl | 10 µl | 1X |
| 10 mM dNTPs | 0.5 µl | 1 µl | 200 µM |
| 10 µM Forward Primer | 1.25 µl | 2.5 µl | 0.5 µM |
| 10 µM Reverse Primer | 1.25 µl | 2.5 µl | 0.5 µM |
| Template DNA | variable | variable | < 1,000 ng |
| Q5 Hot Start High-Fidelity DNA Polymerase | 0.25 µl | 0.5 µl | 0.02 U/µl |
| 5X Q5 High GC Enhancer (optional) | (5 µl) | (10 µl) | (1X) |
| Nuclease-Free Water | to 25 µl | to 50 µl |
Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary and overlay the sample with mineral oil if using a PCR machine without a heated lid.
Transfer PCR tubes to a PCR machine and begin thermocycling:
| A | B | C |
|---|---|---|
| STEP | TEMP | TIME |
| Initial Denaturation | 98°C | 30 seconds |
| 25–35 Cycles | 98°C | 5–10 seconds |
| *50–72°C | 10–30 seconds | |
| 72°C | 20–30 seconds/kb | |
| Final Extension | 72°C | 2 minutes |
| Hold | 4–10°C |
*Use of the NEB Tm Calculator is highly recommended.
