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

    Description

      
    The Q5® Hot Start High-Fidelity 2X Master Mix features a high-fidelity, thermostable, hot start DNA polymerase with 3´→ 5´ exonuclease activity, fused to a processivity-enhancing Sso7d domain to support robust DNA amplification. The addition of an aptamer-based inhibitor allows room temperature reaction setup. With an error rate > 100-fold lower than that of Taq DNA Polymerase and 12-fold lower than that of Pyrococcus furiosus (Pfu) DNA Polymerase, Q5 Hot Start High-Fidelity 2X Master Mix 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 Hot Start High-Fidelity Master Mix contains 2 mM MgCl2. Q5 Hot Start High-Fidelity 2X Master Mix 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.

    Q5-High Fidelity 2X Master Mix formats allow robust amplification of a broad range of targets with a single formulation. 

    M0494
    Amplification of a variety of human genomic amplicons from low to high GC content using Q5 Hot Start High-Fidelity 2X Master Mix. All reactions were set up at room temperature, conducted using 30 cycles of amplification and visualized by microfluidic LabChip® analysis.
      

    Product Source

    An E. coli strain that carries the Q5 High-Fidelity DNA Polymerase gene.

    Advantages and Features

    Applications

    • High-specificity PCR
    • High-fidelity PCR
    • Cloning
    • Long or Difficult Amplification
    • High-throughput PCR

    Properties and Usage

    Storage Temperature

    -20°C

    Heat Inactivation

    No

    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.
    • PCR Amplification (Hot Start, Human Genomic DNA, Master Mix)   :

      The polymerase master mix is tested in a hot start polymerase chain reaction (PCR) using human genomic DNA as the control template and specific primers, resulting in an increase in yield of the expected product and a decrease in non-specific genomic bands when compared to a non-hot start control reaction.

    • PCR Amplification (Master Mix):
      The polymerase master mix is tested in a polymerase chain reaction (PCR) using a control template and specific primers, resulting in the expected product.

    Notes

    1. A precipitate (most noticeable after the first 1–2 freeze/thaw cycles) is not uncommon. To ensure optimal performance, the master mix should be thawed and resuspended prior to use. Stability testing using up to 15 freeze/thaw cycles has shown no negative effect on master mix performance.
    2. Product specifications for individual components in the Q5 Hot Start High-Fidelity 2X Master Mix are available separately.

    References

    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 
      PHOSPHORIBOSYLTRANSFERASE IN ACTIVATION 
      OF THE ANTIVIRAL AGENT T-705 (FAVIPIRAVIR). Molecular Pharmacology Fast Forward. 87247
    3. Hicham Bouabe, Klaus Okkenhaug A Protocol for Construction of Gene Targeting Vectors and Generation of Homologous Recombinant Embryonic Stem Cells. Methods in Molecular Biology .

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, 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 the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What are the advantages to using Q5® Hot Start 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 reaction using Q5® Hot Start 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. There is a precipitate in the bottom of the Master Mix tube? Is this normal?
    8. How do I activate Q5® Hot Start High-Fidelity DNA Polymerase?
    9. 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?
    10. What length of product can be made by Q5® High-Fidelity DNA Polymerase?
    11. I have a tube of the Q5® High GC Enhancer from another product formulation - can I add it to the Q5 Master Mix?
    12. What are the stability and storage requirements of the Q5® Master Mixes?
    13. Does Q5® High-Fidelity DNA Polymerase exhibit a strand displacement activity?
    14. Where can I find help troubleshooting my PCR?
    15. Will Q5® High-Fidelity DNA Polymerase incorporate dUTPs?
    16. I'd like to clone a fragment amplified with Q5® High-Fidelity DNA Polymerase. Do I have to blunt-end clone?
    1. Protocol for Q5® Hot Start High-Fidelity 2X Master Mix
    2. In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)

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