New England Biolabs M0535 Phusion® Hot Start Flex DNA Polymerase
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. Notice to Customers: Phusion® DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. This product is now manufactured by New England Biolabs, Inc. under agreement with, and under the performance specifications of Thermo Fisher Scientific. Further details on this change can be found in the following customer communication. However, it is important to note that the catalog numbers for these products have changed. For any questions, please use our Phusion Request for Technical Support or call NEB Technical Support at 1-800-632-7799. Phusion® is a registered trademark and property of Thermo Fisher Scientific. Source: An E. coli strain that carries the Phusion DNA Polymerase gene. Applications:
DMSO (100 %) MgCl2 solution (50 mM) Phusion® GC Buffer Pack (5X) Phusion® HF Buffer Pack (5X) Enzyme Properties 
Heat Inactivation: No Reaction & Storage Conditions 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. 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 400 μg/ml activated Calf Thymus DNA. Reaction Conditions: 1X Phusion HF or GC Buffer, DNA template, 0.5 μM primers, 200 μM dNTPs (not included), 3% DMSO (optional) and 1 unit of Phusion Hot Start Flex DNA Polymerase in a total reaction volume of 50 μl. Concentration: 2,000 units/ml Storage Conditions: 20 mM Tris-HCl 100 mM KCl 1 mM Dithiothreitol 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol 1 X stabilizers pH 7.4 @ 25°C Storage Temperature: -20°C Notes General notes:
FAQs
Protocols Protocols for Phusion® Hot Start Flex DNA Polymerase Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product. 7.5 kb Genomic DNA PCR: 30 cycles of PCR amplification in a 50 μl reaction containing 50 ng genomic DNA with 1.0 unit of Phusion Hot Start Flex DNA Polymerase in the presence of 200 μM dNTPs and 1.0 μM primers in Phusion HF Buffer results in the expected 7.5 kb product. 20 kb Lambda DNA PCR: 22 cycles of PCR amplification in a 50 μl reaction containing 10 ng Lambda DNA with 1.0 unit of Phusion Hot Start Flex DNA Polymerase in the presence of 200 μM dNTPs and 1.0 μM primers in Phusion HF Buffer results in the expected 20 kb product. Hot Start-Specific Genomic DNA PCR: 25 cycles of PCR amplification in a 25 μl reaction containing 50 ng genomic DNA with 0.5 units of Phusion Hot Start Flex DNA Polymerase in the presence of 200 μM dNTPs and 0.5 μM primers in 1X Phusion GC Buffer results in the expected 665 bp product, free of nonspecific amplification products after pre-incubation at room temperature for 1 hour. Endonuclease Activity: Incubation of a 50 μl reaction in NEBuffer 2 containing a minimum of 10 units of Phusion DNA Polymerase with 200 μM dNTPs and 1 μg of supercoiled ΦX174 DNA for 4 hours at either 37°C or 72°C results in < 10% conversion to the nicked form as determined by agarose gel electrophoresis. References
Reagents Sold Separately Phusion® GC Buffer Pack Phusion® HF Buffer Pack Companion Products Deoxynucleotide Solution Mix Deoxynucleotide Solution Set Detergent-free Phusion® GC Buffer Pack Detergent-free Phusion® HF Buffer Pack Magnesium Chloride (MgCl2) Solution Phusion® GC Buffer Pack Phusion® HF Buffer Pack Phusion® High-Fidelity DNA Polymerase Phusion® High-Fidelity PCR Kit Phusion® High-Fidelity PCR Master Mix with GC Buffer Phusion® High-Fidelity PCR Master Mix with HF Buffer Legal Licenses/Patents/Disclaimers: Phusion® DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. This product is manufactured by New England Biolabs, Inc. under agreement with, and under the performance specifications 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 patents 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. The purchase price of this product includes a limited, non-transferable license under a patent owned by New England Biolabs, Inc., to use this product. No other license under this patent is conveyed expressly or by implication to the purchaser by the purchase of this product. Nucleic acid-based aptamers for use with thermophilic DNA polymerases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. (See Patent Nos. 5,475,096; 5,670,637; 5,696,249; 5,874,557; and 5,693,502). New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the aptamer-based Phusion Hot Start Flex DNA Polymerase for RESEARCH PURPOSES ONLY. Commercial use of the aptamer-based Phusion Hot Start Flex DNA Polymerase requires a license from New England Biolabs, Inc. Please contact busdev@neb.com for more information.
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