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FAQs for Phusion™ Hot Start High-Fidelity DNA Polymerase Protocols for Phusion™ Hot Start High-Fidelity DNA Polymerase FAQs for Polymerases Technical Reference for Polymerases Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE 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 Phusion™ Site-Directed Mutagenesis Kit

Home > Products > Polymerases > Hot Start PCR > Phusion™ Hot Start High-Fidelity DNA Polymerase Phusion™ Hot Start High-Fidelity DNA Polymerase Developed & Manufactured By Finnzymes Oy. Distributed by New England Biolabs, Inc. Catalog # Size Concentration Price Qty   F-540L 500 units 2 units/μl $480.00 F-540S 100 units 2 units/μl $120.00 Prices are in US dollars and valid only for US orders. Download: Manual|MSDS PDF Licensed for PCR Combines very high specificity with the extreme performance of Phusion DNA Polymerase Reduces non-specific amplification and primer degredation Reactions can be set up at room temperature and can be performed with suboptimal primers Description: The unique Phusion™ High-Fidelity DNA Polymerase with a dsDNA-binding domain is now available with a hot start modification. The new Affibody®-based inactivation method of Phusion DNA Polymerase increases the specificity of PCR amplification by preventing non-specific extension of DNA substrate at ambient temperatures prior to the first cycle of PCR. In addition to extreme fidelity, speed and robustness, Phusion Hot Start DNA Polymerase now also delivers extreme specificity to PCR experiments. Phusion Hot Start DNA Polymerase does not require a separate activation step in the PCR protocol. Thus, protocols optimized for Phusion DNA Polymerase can be directly applied to Phusion Hot Start DNA Polymerase reactions. The Phusion™ Technology Incorporating an exciting fusion protein technology, Phusion Hot Start DNA Polymerase brings together a novel Pyrococcus-like enzyme with a processivity-enhancing domain. By fusing a double-strand DNA-binding domain to the polymerase, its processivity can easily be increased 10-fold. Phusion DNA Polymerase exploits this dramatic increase in processivity, resulting in shorter extension times, more robust and high yield amplification, and the ability to amplify long templates in a fraction of the time it takes other polymerases. Figure 1. The structure of Phusion™ High-Fidelity DNA Polymerases. The double-strand DNA-binding domain (purple) is fused to a novel Pyrococcus-like enzyme (green) forming a unique high-performance polymerase - Phusion DNA Polymerase. Extreme fidelity - A New Standard Phusion DNA Polymerases exhibit the lowest error rate thus setting a new standard for high-fidelity PCR. The error rate of Phusion Hot Start DNA Polymerase is equal to Phusion DNA Polymerase, 4.4 x 10-7, determined with a modified lacI-based method. It is approximately 50-fold lower than that of Thermus aquaticus DNA polymerase and 6-fold lower than that of Pyrococcus furiosus DNA polymerase. Figure 2. Relative fidelity values of different DNA polymerases. Fidelity = 1 / error rate. Fidelity assays were done using lacI-based method modified from Frey & Suppmann, 1995. Extreme processivity The Phusion DNA Polymerases have the highest processivity of all thermostable DNA polymerases tested. Table 1. The relative processivity values of Phusion DNA Polymerase and other DNA polymerases. Processivity assay. A 5' FAM-labeled primer was annealed to ssM13mp18 DNA. The primed template was pre-formed in the presence of standard PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 2 mM MgCl2, and 0.1 % Triton®-100) and 200 µM of each dNTPs. DNA polymerase was added to the primed template at a molar ratio of ~1:4,000 to initiate DNA synthesis at 72°C. Samples taken at various times were diluted in gel loading dye, and analyzed on a MJ GeneWorks BaseStation® (MJ Research). The median product length was determined based on the integration of all detectable primer extension products. When the median product length does not change with an increase in reaction time or a decrease in polymerase concentration, it is used as a measure of processivity. Extreme specificity The hot start modification of Phusion Hot Start High-Fidelity DNA Polymerase increases the specificity of PCR amplification by preventing non-specific extension of DNA substrate at ambient temperatures prior to the first cycle of PCR. Extreme speed and yield The fusion of a double-strand DNA-binding domain to Phusion DNA Polymerases multiplies their processivity in respect of other DNA polymerases. This dramatic increase in processivity results in shorter extension times, more robust and high yield amplification, and the ability to amplify long templates in a fraction of the time it takes other polymerases. Extreme robustness The robustness of Phusion Hot Start DNA Polymerase reduces reaction failures and minimizes optimization. Because of its robust performance, Phusion Hot Start High-Fidelity DNA Polymerase is capable of processing templates in the presence of additives such as DMSO, or in reactions containing impurities like debris from cell suspensions. Source: Phusion Hot Start High-Fidelity DNA Polymerase is isolated and purified from an E. coli strain carrying a plasmid with the cloned Phusion DNA Polymerase gene. The Affibody ligand is isolated and purified from an E. coli strain carrying a plasmid with the cloned Affibody encoding gene. Applications: High-fidelity PCR The error rate of Phusion DNA Polymerases is 4.4 x 10-7, which is industry-leading fidelity. Therefore Phusion DNA Polymerase is suitable for all PCR applications requiring great accuracy. Cloning Phusion Hot Start High-Fidelity DNA Polymerase amplifies templates with an accuracy and speed previously unattainable with a single enzyme. This makes it a superior choice for cloning. Phusion DNA Polymerases produce blunt ends to the amplified fragment. Long amplicons Extension and overall cycling times can be significantly reduced when using Phusion Hot Start High-Fidelity DNA Polymerase. Due to the high processivity Phusion DNA Polymerases can amplify long templates in a fraction of the time other polymerases need. Advantages: Extreme specificity Extreme processivity Extreme robustness and yield Reagents Supplied: 5X Phusion™ HF Buffer and 5X Phusion™ GC Buffer DMSO (100 %) MgCl2 solution (50 mM) Reaction & Storage Conditions Unit Definition: One unit is defined as the amount of enzyme that will incorporate 10 nmoles of dNTPs into acid-insoluble form at 74°C in 30 minutes under the stated assay conditions. Concentration: 2 units/μl Storage Conditions: 20 mM Tris-HCl 100 mM KCl 1 mM DTT 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol pH 7.4 @ 25°C Storage Temperature: -20°C Notes General notes: Both Phusion HF Buffer and Phusion GC Buffer contain 7.5 mM MgCl2 in the provided 5X concentration. FAQs Are the DNA fragments produced by Phusion™ Hot Start High-Fidelity DNA Polymerase blunt-ended or do they have the single-base 3´ overhang that Taq DNA Polymerase yields? Does Phusion™ Hot Start High Fidelity DNA Polymerase exhibit a strand displacement activity? I am having trouble amplifying a template that is longer than 5 kb. How can I optimize my product yield using Phusion™ Hot Start High-Fidelity DNA Polymerase? I'd like to clone a fragment amplified wih Phusion™ Hot Start High-Fidelity DNA Polymerase. Do I have to blunt end clone? My template is GC rich or supercoiled. How can I optimize my product yield using Phusion™ Hot Start High-Fidelity DNA Polymerase? What are the advantages to using Phusion™ Hot Start High-Fidelity DNA Polymerase? What is the error rate of Phusion™ Hot Start High-Fidelity DNA Polymerase? What should my primer concentration be when using Phusion™ Hot Start High-Fidelity DNA Polymerase? Why are there high molecular weight smears or DNA in the wells of an agarose gel after a PCR using Phusion™ Hot Start High-Fidelity DNA Polymerase? Why are there low molecular weight discrete bands on an agarose gel after a PCR using Phusion™ Hot Start High-Fidelity DNA Polymerase? Why do I see no product or low yield on an agarose gel after a PCR using Phusion™ Hot Start High-Fidelity DNA Polymerase? Will Phusion™ Hot Start High-Fidelity DNA Polymerase incorporate dUTPs? Are Finnzymes' DNA Polymerases licensed for PCR? References Frey & Suppmann (1995) Biochemica, 2, 34-35. Nord et al. (1997) Nature Biotechnol., 15, 772-777. Wikman et al. (2004) Protein Eng., Des. Sel., 17, 455-462. Chester & Marshak (1993) Anal. Biochem., 209, 284-290. Breslauer et al. (1986) PNAS, 83, 3746-3750. Companion Products 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 Phusion™ Site-Directed Mutagenesis Kit Legal Licenses/Patents/Disclaimers: * PCR license notice: These products are sold under licensing arrangements of Finnzymes Oy with F. Hoffman-La Roche LTD, Roche Molecular Systems, Inc. and Applied Biosystems. The purchase of these products is accompanied by a limited license to use them in the Polymerase Chain Reaction (PCR) process in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front fee, either by payment to Applied Biosystems or as purchased, i.e. an authorized thermal cycler. 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 New England Biolabs, 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.