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Phusion® Hot Start Flex DNA Polymerase
Catalog #
M0535S
M0535L
Size
100 units
500 units
Concentration
2,000 units/ml
2,000 units/ml
Categories: Phusion® High-Fidelity DNA Polymerase Products, Phusion® High-Fidelity DNA Polymerase Products
Applications: Fast PCR, Fast PCR, High-Fidelity PCR | More +
Product
Information
FAQs &
Tech Tips
Protocols &
Manuals
Other Tools &
Resources
Description
Properties and Usage
References
Quality &
Safety
Legal
Information
Advantages and Features
Related Products
Description
High Fidelity DNA Polymerases are important for applications in which the DNA sequence needs to be correct after amplification.
Manufactured and quality-controlled at New England Biolabs, Thermo Scientific® 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.
Reagents Supplied
The following reagents are supplied with this product:
Store at (°C)
MgCl2 solution
Concentration
50 mM
DMSO
100%
Phusion® HF Buffer Pack
-20
5X
Phusion® GC Buffer Pack
-20
5X
Advantages and Features
Features
High fidelity & robustness
High specificity, room temperature reaction setup
Applications
High-specificity PCR
Cloning
Long or Difficult Amplification
High-Throughput PCR
Properties and 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 Temperature
-20°C
Storage Conditions
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.
Related Products
Companion Products
Deoxynucleotide (dNTP) Solution Mix
Deoxynucleotide (dNTP) Solution Set
Detergent-free Phusion® GC Buffer Pack
Detergent-free Phusion® HF Buffer Pack
Magnesium Chloride (MgCl2) Solution
Phusion® High-Fidelity DNA Polymerase
Phusion® High-Fidelity PCR Kit
Phusion® High-Fidelity PCR Master Mix with HF Buffer
Phusion® Hot Start Flex 2X Master Mix
Phusion® High-Fidelity PCR Master Mix with GC Buffer
Materials Sold Separately
Phusion® GC Buffer Pack
Phusion® HF Buffer Pack
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.
FAQs
FAQs
Tech Tips
1. Is Phusion® Hot Start Flex DNA Polymerase the same as Phusion Hot Start DNA Polymerase (#F-540S/L) or Phusion Hot
Start II DNA Polymerase (#F-549S/L), which were previously sold by NEB?
2. Are the DNA fragments products by Phusion® Hot Start Flex DNA Polymerase blunt-ended or do they have the single-base 3’
overhang that Taq DNA Polymerase yields?
3. What are the advantages of using Phusion® Hot Start Flex DNA Polymerase?
4. I am having trouble amplifying a template that is longer than 5kb. How can I optimize my product yield using Phusion® Hot
Start Flex DNA Polymerase?
5. Why do I see no product or low yield on an agarose gel after a PCR using Phusion® Hot Start Flex DNA Polymerase?
6. Will Phusion® Hot Start Flex DNA Polymerase incorporate dUTPs?
7. Why are there low molecular weight discrete bands on an agarose gel after a PCR using Phusion® Hot Start Flex DNA
Polymerase?
8. I'd like to clone a fragment amplified with Phusion® Hot Start Flex DNA Polymerase. Do I have to blunt-end clone?
9. I see foaming when my Phusion® Hot Start Flex-amplified PCR products are spotted on microarray slides.
10. What should my primer concentration be when using Phusion® Hot Start Flex DNA Polymerase?
11. How does the fidelity of Phusion® DNA Polymerase compare to Taq DNA Polymerase?
12. Why are there high molecular weight smears or DNA in the wells of an agarose gel after a PCR using Phusion Hot Start Flex
DNA Polymerase?
13. Does Phusion® Hot Start Flex DNA Polymerase exhibit a strand displacement activity?
14. How do I activate Phusion® Hot Start Flex DNA Polymerase?
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.
Protocols
Datacards
Protocols
1. Protocol for Phusion® Hot Start Flex DNA Polymerase (M0535)
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 [email protected] or fill out the
Technical Support Form for appropriate document.
M0535Datasheet-Lot0011110
M0535Datasheet-Lot0011206
M0535Datasheet-Lot0011209
M0535Datasheet-Lot0011212
M0535Datasheet-Lot0011303
M0535Datasheet-Lot0011306
M0535Datasheet-Lot0021309
M0535Datasheet-Lot0021312
M0535Datasheet-Lot0021406
M0535Datasheet-Lot0021411
M0535Datasheet-Lot0041506
Selection Charts
Usage Guidelines & Tips
Selection Charts
DNA Polymerase Selection Chart
Thermophilic DNA Polymerases
Feature Articles
Feature Articles
Troubleshooting Guides
Polymerase Fidelity: What is it, and what does it mean for your PCR?
Usage Guidelines & Tips
Activity of Restriction Enzymes in a Q5®, Taq or Phusion PCR Mix
Guidelines for PCR Optimization with Thermophilic DNA Polymerases
Troubleshooting Guides
PCR Troubleshooting Guide
Quality Control
Specifications
Datacards
Certificate of Analysis
Safety Data Sheet
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. Further information regarding NEB product quality can be found here.
Endonuclease Activity (Nicking):
The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to
the nicked form is determined by agarose gel electrophoresis.
PCR Amplification (DNA Polymerase):
The polymerase is tested in a polymerase chain reaction (PCR) using a control template and specific primers, resulting in the
expected product.
PCR Amplification (Hot Start, Human Genomic DNA):
The polymerase 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.
Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's
for an individual lot. The following file naming structure is used to name these document files: [Product
Number]_[Size]_[Version]_[Lot Number]
M0535S_L_v1_0041509
M0535S_L_v1_0041602
M0535S_L_v1_0041608
M0535S_L_v1_0061704
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]
M0535S_L_v1
Safety Data Sheet
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
Phusion® Hot Start Flex DNA Polymerase
MgCl2 solution
DMSO
Phusion® HF Buffer Pack
Phusion® GC Buffer Pack
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 [email protected] or fill out the
Technical Support Form for appropriate document.
M0535Datasheet-Lot0011110
M0535Datasheet-Lot0011206
M0535Datasheet-Lot0011209
M0535Datasheet-Lot0011212
M0535Datasheet-Lot0011303
M0535Datasheet-Lot0011306
M0535Datasheet-Lot0021309
M0535Datasheet-Lot0021312
M0535Datasheet-Lot0021406
M0535Datasheet-Lot0021411
M0535Datasheet-Lot0041506
Legal and Disclaimers
Legal and Disclaimers
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc
(NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain
additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at [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.
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.
Disclaimers
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.