Q5® High-Fidelity PCR Kit



Q5® High-Fidelity DNA Polymerase is an ultra 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 Q5 High-Fidelity 2X Master Mix offers robust, high-fidelity performance in a convenient master mix format. 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 MgCl2.

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 Tm Calculator is highly recommended.

This kit contains a sufficient supply of master mix and nuclease-free water to perform 50 polymerase chain reactions (NEB #E0555S) or 200 reactions (NEB #E0555L). For added convenience, gel loading dye, and a Quick-Load DNA LAdder are also included in this kit.


Kit Components

The following reagents are supplied with this product:

Store at (°C)Concentration
Q5® High-Fidelity 2X Master Mix-202X
Nuclease-free Water-20
Quick-Load® Purple 2-Log DNA Ladder (0.1 - 10.0 kb)4
Gel Loading Dye, Purple (6X)256X

Properties and Usage


  1. Product specifications for individual components in the Q5 High-Fidelity PCR Kit are available separately.


  1. Anastassia Voronova Erin Coyne, Ashraf Al Madhoun, Joel V. Fair, Neven Bosiljcic, Catherine St-Louis, Grace Li, Sherry Thurig, Valerie A. Wallace, Nadine Wiper-Bergeron, and Ilona S. Skerjanc. (2013). Hedgehog Signaling Regulates MyoD Expression and Activity. J Biol Chem. 288(6), 4389–4404. PubMedID: PMC3567689
  2. Lieve Naesens, Luke Guddat, Dianne Keough, André B.P. van Kuilenburg, Judith Meijer, Johan Vande Voorde and Jan Balzarini (2013). ROLE OF HUMAN HYPOXANTHINE GUANINE 
    OF THE ANTIVIRAL AGENT T-705 (FAVIPIRAVIR). Molecular Pharmacology Fast Forward. 87247
  3. Hicham Bouabe, Klaus Okkenhaug. (2013). A Protocol for Construction of Gene Targeting Vectors and Generation of Homologous Recombinant Embryonic Stem Cells. Methods in Molecular Biology . 1064, 337-354.


  1. What are the advantages to using Q5® High-Fidelity 2X Master Mix?
  2. What is the fidelity of Q5® High-Fidelity DNA Polymerase?
  3. How should I determine an appropriate annealing temperature for my reaction?
  4. What should my primer concentration be when using Q5® High-Fidelity DNA Polymerase products?
  5. How should I set up a PCR experiment using Q5® High-Fidelity 2X Master Mix?
  6. My template is GC rich or supercoiled. How can I optimize my product yield using Q5® High-Fidelity Master Mixes?
  7. I have a tube of the Q5® High GC Enhancer from another product formulation - can I add it to the Q5 Master Mix?
  8. There is a precipitate in the bottom of the Master Mix tube? Is this normal?
  9. What are the stability and storage requirements of the Q5® PCR Kit?
  10. Are the DNA fragments produced by Q5 High-Fidelity DNA Polymerase blunt-ended or do they have the single-base 3´ overhang that Taq DNA Polymerase yields?
  11. What length of product can be made by Q5® High-Fidelity DNA Polymerase?
  12. Does Q5® High-Fidelity DNA Polymerase exhibit a strand displacement activity?
  13. Will Q5® High-Fidelity DNA Polymerase incorporate dUTPs?
  14. How much of the DNA Ladder should I load on my gel?
  15. Can I use the Quick-Load 2-Lod DNA Ladder to quantify the amount of DNA in my sample?
  16. Where can I find additional help troubleshooting my PCR?


  1. Protocol for a Routine PCR (E0555)
  2. PCR Optimization (E0555)


The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

Selection Charts

Feature Articles

Usage Guidelines & Tips

Interactive Tools

NEB Publications

  • Potapov Vladamir, Ong Jennifer L (2017). Examining Sources of Error in PCR by Single-Molecule Sequencing. PLoS ONE. 12(1): e0169774, PubMedID: 28060945

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • PCR Amplification (Enhancer Dependent, GC-rich):

    The polymerase is tested in a polymerase chain reaction (PCR) using a GC-rich control template and specific primers, resulting in the enhancer-dependent production of the expected product.

Certificate of Analysis

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


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]

Safety Data Sheet

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