Product Class: Other

Phusion® Hot Start Flex DNA Polymerase
recombinant No hifi hotstart pcr 3

Product Introduction

For high speed and high performance PCR. Manufactured and quality-controlled at New England Biolabs, Thermo Scientific® Phusion High-Fidelity DNA Polymerase offers both high fidelity and robust performance.

  • 50X higher fidelity than Taq
  • Robust reactions with minimal optimization
  •  Hot start formulation enables room temperature reaction setup and reduces non-specific amplification
  • High yields with shorter extension times
Catalog # Size Concentration
M0535S 100.0 units 2000 units/ml
M0535L 500.0 units 2000 units/ml

Product Information

Description

High Fidelity DNA Polymerases are important for applications in which the DNA sequence needs to be correct after amplification. Phusion High-Fidelity DNA Polymerase offers both high fidelity and robust performance, and thus can be used for all PCR applications. Its unique structure, a novel Pyrococcus-like enzyme fused with a processivity-enhancing domain, increases fidelity and speed. Phusion DNA Polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. With an error rate 50-fold lower than that of Taq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus DNA Polymerase (1), Phusion is one of the most accurate thermostable polymerases available. Phusion DNA Polymerase possesses 5´→ 3´ polymerase activity, 3´→ 5´ exonuclease activity and will generate blunt-ended products.

Phusion Hot Start Flex DNA Polymerase offers robust, high fidelity performance and room temperature reaction setup. The addition of an aptamer-based inhibitor allows room temperature reaction setup. Phusion Hot Start Flex DNA Polymerase is supplied with standard 5X Phusion HF Buffer, as well as 5X Phusion GC Buffer, which can be used for complex or GC-rich templates. Each of these buffers contains MgCl2 (1.5 mM at the final [1X] reaction concentration). Reactions can also be optimized using the provided DMSO or MgCl2 solutions.

Phusion Hot Start Flex DNA Polymerase is unlike other enzymes and care must be taken when designing cycling protocols. To determine the optimal annealing temperatures for a given set of primers, use of the NEB Tm Calculator is highly recommended.

Product Source

An E. coli strain that carries the Phusion DNA Polymerase gene.
This product is related to the following categories:
Phusion® High-Fidelity DNA Polymerases Products,
This product can be used in the following applications:
Hot Start PCR,
Long Range PCR,
Fast PCR,
High-Fidelity PCR,
Multiplex PCR,
Specialty PCR,
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

Unit Definition

One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74°C.

Storage Buffer

20 mM Tris-HCl
100 mM KCl
1 mM DTT
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
1X stabilizers
pH 7.4 @ 25°C

Heat Inactivation

No

5' - 3' Exonuclease

No

Unit Assay Conditions

25 mM TAPS-HCl (pH 9.3 @ 25°C), 50 mM KCl, 2 mM MgCl2, 1 mM β-mercaptoethanol, 200 μM dNTPs including [3H]- dTTP and 15 nM primed M13 DNA. 

Application Features

  • High-specificity PCR
  • Cloning
  • Long or Difficult Amplification
  • High-Throughput PCR

References

  1. Frey, B. and Suppman, B. (1995). BioChemica. 2, 34-35.
  2. Chester, N. and Marshak, D.R. (1993). Analytical Biochemistry. 209, 284-029.

Protocols, Manuals & Usage

Protocols

  1. Protocol for Phusion® Hot Start Flex DNA Polymerase (M0535)

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. What are the differences between the various available Phusion® products?
  2. My results are not as expected. Where can I find troubleshooting help?
  3. What ends will my PCR products have?
  4. What does exonuclease activity mean for a DNA polymerase?
  5. Does Phusion® High-Fidelity DNA Polymerase exhibit a strand displacement activity?
  6. What are the properties of this polymerase (fidelity, product ends, max amplicon, modified base incorporation, etc.)?
  7. How should I determine the appropriate annealing temperature for my reaction?
  8. What should the final primer concentration be in my PCR?
  9. My template is GC-rich or supercoiled. How can I optimize my product yield using Phusion® High-Fidelity DNA Polymerase?
  10. What are Hot Start and WarmStart® polymerases and when would I use them?

Troubleshooting

Tech Tips

Phusion Hot Start Flex has different annealing temperature requirements than most PCR enzymes.
Please check out the NEB Tm Calculator to help you determine your optimal Phusion annealing temperature.

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

Notice to Customers: Phusion® DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific.

Phusion® is a registered trademark and property of Thermo Fisher Scientific.

Notice to Purchaser: Limited License. The purchase price of this product includes a limited, non-transferable license under U.S. and foreign patent owned by Bio-Rad Laboratories, Inc., to use this product. No other license under these patents is conveyed expressly or by implication to the purchaser by the purchase of this product.

Trademarks

NEW ENGLAND BIOLABS® and Q5® are registered trademarks of New England Biolabs, Inc.

PHUSION® is a registered trademark and property of Thermo Fisher Scientific.
THERMO SCIENTIFIC® is a registered trademark of Thermo Fisher Scientific.

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