Description:
Pfu DNA Polymerase is a highly thermostable DNA polymerase from the hyperthermophilic archaeum Pyrococcus furiosus. The enzyme catalyzes the template-dependent polymerization of nucleotides into duplex DNA in the 5'=>3' direction. Pfu DNA Polymerase also exhibits 3'=>5' exonuclease (proofreading) activity, that enables the polymerase to correct nucleotide incorporation errors. It has no 5'=>3' exonuclease activity.
The main difference between Pfu and alternative enzymes is Pfu's superior thermostability and 'proofreading' properties compared to other thermostable polymerases. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity, resulting in PCR fragments with fewer errors than Taq-generated PCR inserts. Pfu DNA polymerase is efficient for techniques that require high-fidelity DNA synthesis, but can also be used in conjunction with Taq polymerase to obtain the fidelity of Pfu with the speed of Taq polymerase activity.
Applications:
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High-fidelity PCR and primer-extension reactions
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Generation of PCR products for cloning and expression.
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PCR cloning and blunt-end amplification product generation
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RT-PCR for cDNA cloning and expression
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Site-directed mutagenesis
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Blunt-end PCR cloning
Source:
Thermostable DNA polymerase from found in the hyperthermophilic archaeon Pyrococcus furiosus.
Unit Definition:
One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nanomoles of dNTPs into acid insoluble material in 30 minutes at 74°C under standard DNA polymerase assay conditions.
Storage Buffer:
20 mM Tris-HCl, pH 8.0, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.5% Tween 20, and 0.5% Nonidet P40.
Heat Activation:
95% active after 1-hour incubation at 98°C.
Recommended Reaction Conditions:
1X Pfu buffer, 200 µM each dNTP, 0.1-0.5 µM each primer, 2.5 units Pfu DNA polymerase enzyme, 1-100 ng plasmid template DNA, or 100-250 ng genomic template DNA
Recommended Storage Condition:
-20ºC
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