Product Class: Other

Q5®-XT Hot Start High-Fidelity 2X Master Mix

Product Introduction

  • 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)

 Infographic depicting faster amplification with Q5-XT

Bioz Badge Exists : True
Catalog # Size Concentration
M2499S 100 reactions 2 X
M2499L 500 reactions 2 X

Product Information

Description

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.

 

Figure 1: Q5-XT enables amplification on long DNA targets

Gels depicting amplification of long DNA targets, for human genomic DNA and lambda phage DNA

Amplification of specific sequences from human genomic DNA (A) or Lambda phage DNA (B) using Q5-XT High-Fidelity 2X Master Mix. Amplicon sizes are indicated above the gel. Ladder (L) is Quick-Load® 1 kb Extend DNA Ladder (NEB #N3239). All PCR products were visualized by ethidium bromide staining on 1.2% TAE agarose gel.

 

Figure 2: Q5-XT performs well on a broad range of DNA sizes

Comparison of various amplicon lengths for Q5-XT, as compared to other DNA polymerases

PCR assays were performed on six targets with human genomic DNA, utilizing various hot start, high-fidelity PCR master mixes according to each manufacturer's recommended protocols. Ladder (L) is Quick-Load® 100 bp DNA Ladder (NEB #N0467). All PCR products were visualized by ethidium bromide staining on 1.2% TAE agarose gel.

 

Figure 3: Q5-XT is compatible with a universal annealing temperature

Gel depicting amplification with Q5-XT using a universal annealing temperature of 62°C

PCR assays were conducted using a universal thermocycling protocol with a single annealing temperature of 62°C for all nine amplicons regardless of the calculated Ta as obtained using the NEB Tm Calculator. An extension time of 10 seconds was used. All PCR products were visualized by ethidium bromide staining on 1.2% TAE agarose gel. The data highlights that a single annealing temperature and extension time can be used successfully with Q5-XT to support multiple different PCR assays simultaneously, offering significant assay flexibility and convenience. However, when the experimental annealing temperature deviates from the optimal primer annealing temperature, reduced product yield (i.e., reaction efficiency) or poor specificity may occur. As a result, when trying to achieve the best performance in PCR, we recommend using the optimal annealing temperature as determined by a temperature gradient or Tm Calculator. However, when convenience is desired or necessary, a single annealing temperature for multiple different primer sets can be used. Learn more in our application note. Ladder (L) is Quick-Load® 100 bp DNA Ladder (NEB #N0467).

 

Figure 4: Q5-XT supports robust DNA amplification on a broad range of GC content

Q5-XT Master Mix GC content coverage, with the recommended inclusion of GC enhancer

For targets over 65% GC content, the addition of 5X Q5 GC Enhancer, available as a component of Q5 Reaction Buffer Pack (NEB #B9027), is 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

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • NEB # Stored at (°C)
    M2499S -20
    Component Name Component # Stored at (°C) Amount Concentration
    Q5®-XT Hot Start High-Fidelity 2X Master Mix M2499SVIAL -20 2 x 1.25 ml 2 X
  • NEB # Stored at (°C)
    M2499L -20
    Component Name Component # Stored at (°C) Amount Concentration
    Q5®-XT Hot Start High-Fidelity 2X Master Mix M2499SVIAL -20 10 x 1.25 ml 2 X

Properties & Usage

Materials Required but not Supplied

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

Tools & Resources

Selection Charts

Web Tools

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?

Troubleshooting

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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