Product Class: Other

NEBNext® Q5® Hot Start HiFi PCR Master Mix
cloned at NEB recombinant No

Product Introduction

NEBNext® Q5 Hot Start HiFi PCR Master Mix is the original hot start NEBNext formulation of Q5 High-Fidelity DNA polymerase. This formulation is also included as a component in all of the original NEBNext Ultra kits, for DNA and RNA library preparation.

However, the NEBNext Ultra II Q5 Master Mix (NEB #M0544) is now available, and this more recent hot start formulation further improves the uniformity of amplification of libraries, including superior performance with GC-rich regions. The NEBNext Ultra II Q5 Master Mix is also included as a component in all NEBNext Ultra II kits, for DNA and RNA library preparation.
Catalog # Size Concentration
M0543S 50.0 reactions
M0543L 250.0 reactions

Product Information

Description






 

NEBNext® Q5 Hot Start HiFi PCR Master Mix is the original hot start NEBNext formulation of Q5 DNA polymerase. This formulation is also included as a component in all of the original NEBNext Ultra kits, for DNA and RNA library preparation.

The NEBNext Ultra II Q5 Master Mix (NEB #M0544) is now available, which further improves the uniformity of amplification of libraries, including superior performance with GC-rich regions.

The polymerase component of both master mixes, Q5 High-Fidelity DNA Polymerase, is a novel thermostable DNA polymerase that possesses 3´→5´ exonuclease activity, and is fused to a processivity-enhancing Sso7d domain. Q5 also has the highest fidelity available (> 280-fold higher than that of Taq DNA Polymerase), resulting in ultra-low error rates.

This convenient 2X master mix contains dNTPs, Mg++ and a proprietary buffer, and requires only the addition of primers and DNA template for robust amplification. The inclusion of the hot start aptamer allows convenient room temperature reaction set up.

Depth of coverage correlation with unamplified libraries
Depth of coverage correlation with unamplified libraries
Average depth of coverage of each 10 kb window of the hg19 reference genome is plotted for NEBNext Q5 Hot Start HiFi PCR Master Mix on the Y axis, and correlated with the number of reads from a library prepared without an amplification step (PCR-Free) on the X axis. Libraries were prepared using the NEBNext Ultra DNA Library Prep Kit (NEB #E7370), sequenced on a HiSeq® 2500 mapped to the hg19 reference genome (Bowtie 2.1.0), duplicate marked (Picard 1.56), and downsampled to 420M reads (Picard 1.56). Reads on each 10 kb interval of hg19 were counted using Bedtools (2.19.1), and average coverage was calculated by dividing read bases the interval length (10 kb).

NEBNext Q5 Hot Start HiFi PCR Master Mix minimizes GC bias
NEBNext Q5 Hot Start HiFi PCR Master Mix minimizes GC bias
Libraries of human (IMR-90) or E. coli K12 genomic DNA were unamplified (“PCR-free”) or amplified using NEBNext Q5 Hot Start HiFi PCR Master Mix, and sequenced on an Illumina MiSeq®. GC bias plots were generated, with %GC content of 100 bp windows on the X axis. Normalized coverage is indicated by the blue circles, %GC of the reference sequence indicated by the red lines and base quality at %GC indicated by the green line. Similar GC bias was observed for the libraries amplified with NEBNext Hot Start HiFi PCR Master Mix compared to PCR-free libraries.

Reaction Conditions
NEBNext Q5 Hot Start HiFi PCR Master Mix, DNA template and 0.5 μM to 1.25 μM primers (depending on sample input) in a total reaction volume of 50 μl.

Product Source

An E. coli strain that carries the Q5 DNA Polymerase gene.
This product is related to the following categories:
Q5® High-Fidelity DNA Polymerases Products,
Master Mixes Products,
Next Generation Sequencing Library Preparation Products,
This product can be used in the following applications:
Polymerases for NGS Library Preparation,
Hot Start PCR,
Fast PCR,
Specialty PCR,
DNA Amplification, PCR & qPCR,
Routine PCR,
PCR

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Heat Inactivation

No

Application Features

  • Next generation sequencing library amplification
  • High-fidelity PCR
  • Difficult amplification
  • High-throughput PCR

Product Notes

  1. To ensure optimal performance, themaster mix should be thawed and resuspendedprior to use. Stability testing using up to 30 freeze/thaw cycles has shown no negative effect onmaster mix performance. The NEBNext Q5 HotStart HiFi PCR Master Mix may be liquid at –20°C.

Protocols, Manuals & Usage

Protocols

  1. Protocol for a PCR reaction using NEBNext® Q5® Hot Start HiFi PCR Master Mix (M0543)

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. What’s New About NEBNext® Q5 Hot Start HiFi PCR Master Mix?
  2. What types of beads can be used with NEBNext® Q5 Hot Start HiFi PCR Master Mix?
  3. Is NEBNext® Q5 Hot Start HiFi PCR Master Mix the same as the Q5® Hot Start High-Fidelity 2X Master Mix (M0494)?
  4. Is NEBNext® Q5 Hot Start HiFi PCR Master Mix the same as NEBNext® High-Fidelity 2X PCR Master Mix (M0541)?
  5. Is NEBNext® Q5 Hot Start HiFi PCR Master Mix the same as NEBNext® High-Fidelity 2X PCR Master Mix (M0541), or Q5 Hot Start High-Fidelity 2X PCR Master Mix (M0494)?
  6. Do I need to make any changes to the cycling conditions for my NGS library amplification I have previously been using with Phusion High-Fidelity PCR Master Mix?
  7. How many cycles of PCR should I perform with NEBNext® Q5 Hot Start HiFi PCR Master Mix?
  8. Are the DNA products produced by NEBNext® Q5 Hot Start HiFi PCR Master Mix blunt-ended or do they have a single base 3’ overhang?
  9. What do I do if I see precipitation in the NEBNext Q5 Hot Start HiFi PCR Master Mix?
  10. What are the advantages of using NEBNext® Q5 Hot Start HiFi PCR Master Mix?
  11. Will NEBNext® Q5 Hot Start HiFi PCR Master Mix incorporate dUTPs?

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.

Licenses

This product is covered by one or more patents.

This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows.

For additional information or to inquire about commercial use, please contact busdev@neb.com.

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