PCR with Q5® Polymerase (M0491)
New England Biolabs
Published: 2022-02-19 DOI: 10.17504/protocols.io.be6bjhan
Abstract
This protocol describes methods for PCR using Q5® High-Fidelity DNA Polymerase (M0491).
Before start
Please note that protocols with Q5® High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.
Steps
1. All components should be mixed prior to use. Q5 High-Fidelity DNA Polymerase may be diluted in 1X Q5 Reaction Buffer just prior to use in order to reduce pipetting errors.
Set up the following reaction On ice:
Note
| 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 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 |
2.
Gently mix the reaction.
3.
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.
4.
Quickly transfer PCR tubes to a PCR machine preheated to the denaturation temperature (98°C) and begin thermocycling.
Thermocycling Conditions for a Routine PCR:
| 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.
