• Get precise results with Q5's high fidelity (~280x Taq)
• Reduce turnaround time with fast cycling conditions (5–15 seconds/kb extension times)
• Optimized formulation enables robust, reliable amplification of diverse targets (from high AT to high GC) up to 20 kb and supports amplification of simple templates up to 30 kb
• Improve reaction specificity with a hot start aptamer that allows convenient room temperature setup
• Compatible with a 62°C universal annealing temperature protocol. Learn more in our application note
• Also available: Q5 standalone polymerase (NEB #M0491, NEB #M0493, NEB #M0515) and master mix formats (NEB #M0492, NEB #M0494, NEB #M0578, NEB #M0580)

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Product Information

When DNA targets are long or complex, or when speed is a priority, Q5-XT Hot Start High-Fidelity 2X Master Mix provides a faster, more robust amplification solution while maintaining the performance you expect of Q5. With Q5-XT, spend less time troubleshooting, have confidence in the accuracy of your amplicon, and accelerate your workflows.

The Q5-XT Hot Start High-Fidelity 2X Master Mix contains Q5 DNA Polymerase, a high-fidelity, thermostable enzyme with 3′→5′ exonuclease activity, fused to an Sso7d-derived domain to enhance processivity. Its optimized buffer system delivers high yields when amplifying long targets, strong performance on short targets, and fast thermocycling with extension times of 5–15 seconds per kb. An aptamer-based hot start inhibitor enables convenient room-temperature setup while reducing non-specific amplification.

With an error rate approximately 280-fold lower than Taq DNA Polymerase, Q5 is well suited for applications that require exceptionally accurate DNA amplification. The 2X master mix includes dNTPs, Mg++, and a broadly compatible buffer designed to support robust amplification across a wide range of AT and GC content. At a 1X working concentration, Q5-XT provides 1.6 mM Mg++.

Q5-XT is compatible with a 62 °C universal annealing temperature. For optimal results and performance, primer-specific annealing temperatures are recommended. To determine the optimal annealing temperatures for a given set of primers, use of the NEB Tm Calculator is highly recommended.

This product is related to the following categories:

• Q5® High-Fidelity DNA Polymerases
• Master Mixes

This product can be used in the following applications:

• Hot Start PCR
• Long Range PCR
• Fast PCR
• High-Fidelity PCR
• Multiplex PCR
• DNA Amplification, PCR & qPCR
• Routine PCR

Reagents Supplied

The following reagents are supplied with this product:

Properties & Usage

Materials Required but not Supplied

• Template DNA and associated forward and reverse primers
• Nuclease-free Water (NEB #B1500)

Protocols, Manuals & Usage

Protocols for this product

1. PCR using Q5®-XT Hot Start High-Fidelity 2X Master Mix (NEB #M2499)

Application Notes

• Universal Annealing Temperature in PCR and its Impact on Amplification Results

Tools & Resources

Selection Charts

• DNA Polymerase Selection Chart

Web Tools

• Tm Calculator

FAQs & Troubleshooting

FAQs

1. What are the advantages of using Q5® High-Fidelity DNA Polymerase?
2. What are the differences between the numerous Q5® Polymerase products available?
3. My PCR results are not as expected. Where can I find troubleshooting help?
4. What ends will my PCR products have?
5. What does exonuclease activity mean for a DNA polymerase?
6. Does Q5® High-Fidelity DNA Polymerase exhibit a strand displacement activity?
7. What are the properties of this polymerase (fidelity, product ends, max amplicon, modified base incorporation, etc.)?
8. How should I determine the appropriate annealing temperature for my reaction?
9. What should the final primer concentration be in my PCR?
10. When and how should I use the Q5® High GC Enhancer?
11. What are Hot Start and WarmStart® polymerases and when would I use them?