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FAQs for Phusion™ High-Fidelity DNA Polymerase Protocols for Phusion™ High-Fidelity DNA Polymerase FAQs for Polymerases Technical Reference for Polymerases Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder PCR Selection Tool Isoschizomers DNA Sequences and Maps REBASE Detergent-free Phusion™ GC Buffer Pack Detergent-free Phusion™ HF Buffer Pack Phusion™ GC Buffer Pack Phusion™ HF Buffer Pack Phusion™ High-Fidelity PCR Kit Phusion™ High-Fidelity PCR Master Mix with GC Buffer Phusion™ High-Fidelity PCR Master Mix with HF Buffer Phusion™ Hot Start High-Fidelity DNA Polymerase Phusion™ Site-Directed Mutagenesis Kit

Home > Products > Polymerases > High-Fidelity PCR > Phusion™ High-Fidelity DNA Polymerase Phusion™ High-Fidelity DNA Polymerase Developed & Manufactured By Finnzymes Oy. Distributed by New England Biolabs, Inc. Catalog # Size Concentration Price Qty   F-530L 500 units 2 units/μl $412.00 F-530S 100 units 2 units/μl $103.00 Prices are in US dollars and valid only for US orders. Download: Manual|MSDS PDF Licensed for PCR* Highest fidelity of any commercial thermostable polymerase (50X greater than Taq) Industry leading processivity is 10X higher than Pfu and 2X higher than Taq Unrivalled robustness and yield with minimal amounts of enzyme and little optimization Dramatically reduced extension times (5X faster than Pfu and 2X faster than Taq) Description: The unique Phusion™ High-Fidelity DNA Polymerase offers performance that no other enzyme can match. The Phusion DNA Polymerase generates templates with an accuracy and speed previously unattainable with a single enzyme, even on your most difficult templates. The unique structure and characteristics of this polymerase make Phusion DNA Polymerase a superior choice for cloning, and sets a new standard for PCR performance: its error rate is 50-fold lower than that of Thermus aquaticus, and 6-fold lower than that of Pyrococcus furiosus - making it the most accurate of available thermostable polymerases. Phusion DNA Polymerase possesses processivity 10-fold greater than Pyrococcus furiosus and twice that of Thermus aquaticus. The Phusion Technology: Incorporating an exciting new fusion protein technology developed by Finnzymes Oy in collaboration with MJ Bioworks, Inc, Phusion High-Fidelity 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 High-Fidelity DNA Polymerase exploits this dramatic increase in processivity, resulting in shorter extension times, more robust and high yield amplification, and the ability to copy long templates in a fraction of time. Figure 1: The depiction of Phusion High-Fidelity DNA Polymerase. The double-stranded DNA binding domain (blue) is fused with a novel Pyrococcus-like enzyme (green) forming a unique high performance polymerase. Figure 2: Error rates of different DNA polymerases. Fidelity assays were done using lac I-based method modified from Frey & Suppmann, 1995. Extreme fidelity: Using a lac I-based method modified from previous studies, the error rate is determined to be 4.4 x 10-7 in Phusion HF Buffer, which is approximately 50-fold lower than that of Thermus aquaticus, and 6-fold lower than Pyrococcus furiosus. Phusion High-Fidelity DNA Polymerase exhibits the lowest error rate thus making it the new standard for high-fidelity PCR. Table 1: The relative processivity values of Phusion 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 ~1:4000 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 processivity: The Phusion polymerase has the highest processivity of all thermostable DNA polymerases tested. Figure 3: A 3.8 kb human beta globin gene was amplified according to supplier's recommendations using varying extension times. Extreme speed and yield: The fusion of a double-strand DNA binding domain to Phusion High-Fidelity DNA Polymerase multiplies its 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. A 3.8 kb human genomic DNA fragment was amplified with various polymerases using extension times ranging 1-7 min. 40 sec. Phusion High-Fidelity DNA Polymerase gave strong specific bands even with the shortest extension time, completing the 3.8 kb fragment with only a 1 min. combined annealing and extension step. Enzyme amounts also indicate that significantly less of the Phusion High-Fidelity DNA Polymerase is required. Figure 4: A random set of 16 clones from a Thermus sp. genomic library was amplified from bacterial colonies. The amplicon size varied between 1-10 kb. All 16 amplicons were amplified in Phusion HF Buffer using the same reaction conditions according to supplier's instruction. Extreme robustness: The random amplification of complex genomic Thermus species library illustrates the robust performance of Phusion High-Fidelity DNA Polymerase. Phusion DNA Polymerase amplified 15 of the 16 randomly selected amplicons (94%) with high yields. The success rate of Pyrococcus furiosus was 56% and Thermus aquaticus 62% with noticeably lower yields. Because of its robust performance, Phusion 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: Isolated and purified from an E. coli strain carrying a plasmid with the cloned Phusion DNA Polymerase gene. Applications: High-fidelity PCR The error rate of Phusion High-Fidelity DNA Polymerase 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 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 Polymerase produces blunt ends to the amplified fragment. Long amplicons Extension and overall cycling times can be significantly reduced when using Phusion High-Fidelity DNA Polymerase. Due to the high processivity Phusion DNA Polymerase can amplify long templates in a fraction of the time other polymerases need. DHPLC PCR products used for DHPLC analysis are recommended to be free of detergents. Detergent-free reaction buffers are available for Phusion High-Fidelity DNA Polymerase. Microarray Detergents can cause foaming when PCR products are spotted on microarray slides. Detergent-free reaction buffers are available for Phusion High-Fidelity DNA Polymerase. Advantages: Extreme fidelity Extreme processivity Extreme speed and yield Extreme robustness Reagents Supplied: 5X Phusion™ HF Buffer and 5X Phusion™ GC Buffer DMSO (100 %) MgCl2 solution (50 mM) Reaction & Storage 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 0.5% Tween-20 0.5% Nonidet P40 pH 7.4 @ 25°C Storage Temperature: -20°C Stable for at least one year from the assay date. 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™ High-Fidelity DNA Polymerase/Phusion™ High-Fidelity PCR Master Mix blunt-ended or do they have the single-base 3´ overhang that Taq DNA Polymerase yields? My template is GC rich or supercoiled. How can I optimize my product yield using Phusion™ High-Fidelity DNA Polymerase? What are the advantages to using Phusion™ High-Fidelity DNA Polymerase? What is the error rate of Phusion™ High-Fidelity DNA Polymerase? I am having trouble amplifying a template that is longer than 5kb. How can I optimize my product yield using Phusion™ High-Fidelity DNA Polymerase? Why do I see no product or low yield on an agarose gel after a PCR using Phusion™ 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™ High-Fidelity DNA Polymerase? Will Phusion™ High-Fidelity DNA Polymerase incorporate dUTPs? Why are there low molecular weight discrete bands on an agarose gel after a PCR using Phusion™ High-Fidelity DNA Polymerase? I'd like to clone a fragment amplified with Phusion™ High-Fidelity DNA Polymerase/Phusion™ High-Fidelity PCR Master Mix. Do I have to blunt end clone? I see foaming when my Phusion™ DNA Polymerase amplified PCR products are spotted on microarray slides. What should my primer concentration be when using Phusion™ High-Fidelity DNA Polymerase/Phusion™ High-Fideltiy PCR Master Mix? Does Phusion High-Fidelity DNA Polymerase exhibit a strand displacement activity? Are Finnzymes' DNA Polymerases licensed for PCR? Can Phire DNA Polymerase or Phusion High-Fidelity DNA Polymerase be used in Crimson Taq Buffer ? Is Phusion High-Fidelity DNA Polymerase stable at room temperature? Companion Products Detergent-free Phusion™ GC Buffer Pack Detergent-free Phusion™ HF Buffer Pack Phusion™ GC Buffer Pack Phusion™ HF Buffer Pack Phusion™ High-Fidelity PCR Kit Phusion™ High-Fidelity PCR Master Mix with GC Buffer Phusion™ High-Fidelity PCR Master Mix with HF Buffer Phusion™ Hot Start High-Fidelity DNA Polymerase 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.