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
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
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
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
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
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
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).
This product is related to the following categories:
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 & Change Notifications
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]
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]
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.
Based on your Freezer Program type, you are trying to add a product to your cart that is either not allowed or not allowed with the existing contents of your cart. Please review and update your order accordingly. If you have any questions, please contact Customer Support at freezers@neb.com or 1-800-632-5227 x 8.
Choose your country
North America
Europe
Asia-Pacific
Session Expired
Your session has expired
You have been idle for more than 20 minutes, for your security you have been logged out. Please sign back in to continue your session.
Please sign in to continue shopping.
Institution Changed
Your profile has been mapped to an Institution, please sign back for your profile updates to be completed.
Sign in to your NEB account
To save your cart and view previous orders, sign in to your NEB account. Adding products to your cart without being signed in will result in a loss of your cart when you do sign in or leave the site.
![[Duck photo] Learn more about the Q5® DNA Polymerase family of products](https://media.neb.com/asset/eebadfa7-45f3-419d-9c75-5c05a26bb921/Webp_JPG_PSD/Q5_LearnMore_Banner.webp?=)
![[Icon] Use our Tm Calculator to determine your primers' optimal annealing temperature.](https://media.neb.com/asset/46e6c17d-deda-4972-a20c-2c3463d03d8b/Webp_JPG_PSD/TmCalculator_Banner.webp?=)
Powered by Bioz
