Pfu DNA Ploymerase produced by our company is a recombinant protein expressed from cloned Escherichia coli with Pyrococcusfuriosis DNA Ploymerase gene and isolated by column purification, with a molecular weight of 90kDa. Because Pfu enzyme has 3 '→5' exonuclease activity, it can correct the mismatch caused by DNA amplification, but the traditional Taq enzyme cannot. Other heat-resistant DNA Polymerase such as Vent, Deep Vent, Tli, UITma, etc., although it has the correction function. But the Pfu enzyme has the lowest error rate of any heat-resistant DNA Polymerase found so far. Pfu enzyme has no 5 '→3' exonuclide activity, and the elongation rate in PCR reaction is generally 0.5-1kb/min, which is lower than Taq enzyme. Pfu enzyme has better thermal stability than Taq enzyme, and still maintains more than 90% activity at 95℃ for 1h. For templates with high GC content, denaturation temperature can be increased to 98℃ without affecting its activity. The PCR product of Pfu enzyme is flat-terminal, which can be treated with A and then connected with T-vector or flat-terminal cloning vector. It is generally used for high-fidelity DNA amplification, such as gene expression cloning, gene site-specific mutation, intracellular gene point mutation analysis (SNP), and terminal complement equality.

Pfu Source: Escherichia coli expressing Pfu DNA polymerase.

Pfu enzyme storage buffer: 50mM Tris-HCl, pH 8.2; 0.1mM EDTA; 1mM DTT; 0.1% Nonidet P40; 0.1% Tween 20; 50% glycerin.

Pfu unit definition: under the condition of 74℃, the amount of enzyme required to catalyze the incorporation reaction of 10nmmol dNTP into acid insoluble substances within 30min is one unit.

Pfu use:

1) High-fidelity DNA amplification in vitro by conventional PCR reaction;

2) Gene site-specific mutation and splicing.

Pfu Precautions:

1. Enzyme concentration: It is recommended to use 1.25 units of Pfu DNA polymerase in a 50mL system, because the enzyme has 3 '-5' exonuclease activity, and high concentrations of the enzyme may increase the possibility of degrading the primer. It is best to add Pfu DNA polymerase after adding dNTP. The various components of the PCR reaction were mixed in an ice bath.

2, Elongation time: the elongation rate of Pfu DNA polymerase is lower than that of Taq DNA polymerase, about 600 bases per minute (Taq DNA polymerase is about 2000 bases per minute), so it is recommended to amplify 1Kb of DNA with 2min elongation time. For most reactions, 25 to 35 amplification cycles are sufficient.

Pfu storage: Store at -20℃ for at least one year.

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