Product Class: Other

Q5 Quick-Load™ Hot Start High-Fidelity 2X Master Mix


Catalog #M0580

Product Introduction

  • High fidelity amplification in a convenient format
  • Master mix includes Q5® Hot Start High-Fidelity DNA Polymerase, dNTPs, and Mg++ in a broad-use buffer
  • Superior performance for a wide range of amplicons (from high AT to high GC)
  • Hot start aptamer offers increased reaction specificity with the benefit of room temperature setup
  • Includes inert tracking dyes for easy and direct loading of PCR products onto gels
  • Also available: Q5 standalone polymerase (NEB #M0491, NEB #M0493) and colorless master mix formats (NEB #M0492, NEB #M0494)

Product Information

Description

[Duck photo] Learn more about the Q5® DNA Polymerase family of products


The Q5 Quick-Load Hot Start High-Fidelity 2X Master Mix offers high-fidelity performance in a convenient master mix format. The master mix features Q5, a high-fidelity, thermostable DNA polymerase with 3' to 5´ exonuclease activity fused to a processivity-enhancing Sso7d domain. With an error rate ~280-fold lower than that of Taq DNA Polymerase, Q5 offers highly accurate amplification and can be used to amplify difficult or long targets (up to 20 kb for simple templates). The master mix includes hot start polymerase, dNTPs, Mg++, and two commonly used tracking dyes for easy and direct loading of PCR products onto gels. On a 1% agarose gel in 1X TBE, Xylene Cyanol FF migrates at ~3-5 kb and Tartrazine migrates at ~50 bp. The broad-use buffer requires only the addition of primers and DNA template for robust amplification, regardless of GC content.

The addition of a hot start aptamer offers increased reaction specificity and enables room temperature reaction setup. The aptamer-based inhibition/activation process is fully reversible. At the end of thermal cycling, when the temperature of the reaction is decreased, the aptamer rebinds to the enzyme, inhibiting any further activity in the sample.

To determine the optimal annealing temperature for a given set of primers, use of the NEB Tm Calculator is highly recommended.


Figure 1: Q5 formats support robust amplification of a broad range of targets

Coverage diagram of Q5 High-Fidelity DNA Polymerases, with options for flexibility or convenience
For flexibility, standalone Q5 polymerase products and the included High GC Enhancer can support amplification of targets with differing amounts of GC content. For convenience, and to amplify a broad range of target GC content, Q5 master mixes are recommended.

Figure 2: Migration of tracking dyes contained in Quick-Load® PCR reagents during electrophoresis

Green visible tracking dye with Quick-Load® separates to blue ~ 3-5 kb and yellow ~ 50 bp
Quick-Load PCR reagents enable direct loading of PCR products onto agarose DNA gels for analysis. Quick-Load amplification products deliver the same performance as their non-Quick-Load equivalents, while incorporating two PCR-inert tracking dyes for enhanced visualization. During gel electrophoresis, the blue Xylene Cyanol FF dye co-migrates with DNA fragments of approximately 3-5 kb in a 1% agarose gel, while the yellow Tartrazine dye migrates at approximately 50 bp for clear monitoring of both large and small DNA fragments. Additionally, the combination of the blue and yellow dyes results in a green reagent that enables visual tracking of reaction set up.

Figure 3: Q5 master mix products can amplify long targets with high specificity

Gel depicting amplification of long amplicons with Q5 Quick-Load Hot Start High-Fidelity DNA Polymerase
Triplicate PCR reactions were performed using two 5 kb human targets with 10 ng of human genomic DNA, utilizing various hot-start, high-fidelity PCR master mixes according to each manufacturer's recommended protocols. Both Q5 Quick-Load™ Hot Start High-Fidelity 2X Master Mix and Q5® Hot Start High-Fidelity 2X Master Mix demonstrated robust and highly specific amplification of long amplicons. In contrast, the presence of multiple bands in other master mixes indicates non-specific amplification. The Quick-Load® Purple 1 kb DNA Ladder (NEB #N0552) was used as a molecular size marker.

Figure 4: Q5 master mix products are capable of amplifying GC-rich targets

Gel depicting amplification of GC-rich targets with Q5 Quick-Load HotStart High-Fidelity DNA Polymerase
Triplicate PCR reactions were performed using 4 human targets with a high GC content. The amplification was performed with 10 ng of human genomic DNA, utilizing various hot-start, high-fidelity PCR master mixes according to each manufacturer's recommended protocols. Both Q5 Quick-Load™ Hot Start High-Fidelity 2X Master Mix and Q5® Hot Start High-Fidelity 2X Master Mix consistently demonstrated robust and highly specific amplification of GC-rich amplicons. L = Quick-Load® Purple 100 bp DNA Ladder (NEB #N0551).

Figure 5: Q5 master mix products exhibit robust amplification on a broad range of amplicons

Gel depicting robust amplification of targets of different lengths and different GC content with Q5 Quick-Load Hot Start High-Fidelity DNA Polymerase
Triplicate PCR reactions were performed using 4 human targets with average length and GC content. The amplification was performed with 10 ng of human genomic DNA, utilizing various hot-start, high-fidelity PCR master mixes according to each manufacturer's recommended protocols. Both Q5® Hot Start High-Fidelity 2X Master Mix and Q5 Quick-Load™ Hot Start High-Fidelity 2X Master Mix consistently demonstrated robust and highly specific amplification across a range of GC content. L = Quick-Load® Purple 100 bp DNA Ladder (NEB #N0551).

Figure 6: Q5 master mix products are stable up to 24 hours at room temperature

Gel depicting room-temperature stability with Q5 Quick-Load Hot Start High-Fidelity DNA Polymerase
Q5® Hot Start High-Fidelity 2X Master Mix and Q5 Quick-Load™ Hot Start High-Fidelity 2X Master Mix ensure the stability of assembled reactions at room temperature for up to 24 hours, enabling efficient high-throughput PCR using liquid-handling systems. To evaluate stability, a 562 bp target was amplified from 10 ng of human genomic DNA using various hot-start, high-fidelity PCR master mixes, following each manufacturer’s recommended protocols. Reactions were either immediately loaded onto a thermal cycler (0 hr) or held at room temperature for 24 hours prior to cycling (24 hr). L = Quick-Load® Purple 100 bp DNA Ladder (NEB #N0551).

[Icon] Use our Tm Calculator to determine your primers' optimal annealing temperature.

This product is related to the following categories:
Q5® High-Fidelity DNA Polymerases,
Master Mixes,
This product can be used in the following applications:
High-Fidelity PCR,
DNA Amplification, PCR & qPCR

Properties & Usage

Materials Required but not Supplied

  • Thermocycler
  • PCR tubes
  • Mineral oil (if needed)

Protocols, Manuals & Usage

Protocols

  1. PCR Using Q5 Quick-Load™ Hot Start High-Fidelity 2X Master Mix (NEB #M0580)

Application Notes

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 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. What are the properties of this polymerase (fidelity, product ends, max amplicon, modified base incorporation, etc.)?
  7. Does Q5® High-Fidelity DNA Polymerase exhibit a strand displacement activity?
  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. There is a precipitate in the bottom of the buffer tube. Is this normal?
  12. What are Hot Start and WarmStart® polymerases and when would I use them?
  13. What is the advantage of using Q5 Quick-Load™?
  14. What dyes are present in Q5 Quick-Load™?
  15. Do the tracking dyes present in Q5 Quick-Load™ interfere with downstream applications?

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]

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 [email protected].

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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