PCR with Q5® High-Fidelity 2X Master Mix - CHEM 384/584
New England Biolabs
Abstract
The Q5 High-Fidelity 2X Master Mix offers robust, high-fidelity performance in a convenient master mix format. The Q5 High- Fidelity 2X Master Mix features a high-fidelity, thermostable DNA polymerase with 3´→ 5´ exonuclease activity, fused to a processivity-enhancing Sso7d domain to support robust DNA amplification. With an error rate ~280-fold lower than that of Taq DNA Polymerase, Q5 High-Fidelity DNA Polymerase is ideal for cloning and can be used for long or difficult amplicons. The convenient master mix formulation is supplied at a 2X concentration. The mix contains dNTPs, Mg++and a proprietary broad-use buffer requiring only the addition of primers and DNA template for robust amplification regardless of GC content. When used at the recommended 1X final concentration, the Q5 High-Fidelity Master Mix contains 2 mM Mg++. Q5 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 TmCalculatormCalculator is highly recommended.
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
Set up the following reaction On ice:
| A | B | C | D |
|---|---|---|---|
| Component | 25 µl Reaction | 50 µl Reaction | Final Concentration |
| Q5 High-Fidelity 2X Master Mix | 12.5 µl | 25 µl | 1X |
| 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 |
| 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.
Quickly transfer PCR tubes to a preheated (98°C) PCR machine 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 CalculatorTm Calculator is highly recommended.
