90 kDa (Reducing)
Store at -25 ~ -15℃ for 2 years
[1] Picard Véronique, et al. "A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase." Nucleic Acids Research 22.13(1994):2587-91.
[2] Yan, Wang , et al. "A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro." Nucleic Acids Research 3(2004):1197.
Pfu DNA Polymerase is a thermostable enzyme that replicates DNA at 75°C. It catalyzes the polymerization of nucleotides into duplex DNA in the 5´→3´ direction in the presence of magnesium. The enzyme has a molecular weight of approximately 90,000 daltons as estimated from the predicted amino acid sequence and exhibits 3´→5´ exonuclease (proofreading) activity. Pfu DNA Polymerase is recommended for use in PCR and primer extension reactions that require high fidelity.
Storage Solution : 5 U/ul Pfu DNA Polymerase, 20 mM Tris-HCl, 0.1 mM EDTA, 0.1% Tween20, 0.1% triton X100, 1 mM DTT, 100 mM KCl, 50%Glycerol、pH 8.2 @ 25°C
10* Reaction Buffer: 200 mM Tris-HCl (pH 8.8) , 20 mM MgSO4, 100 mM KCl, 100 mM (NH4) 2SO4, 1% Triton X-100, 1 mg/ml nuclease-free BSA
1. Set up a 50 μl PCR reaction system as follows:
Reagent | Volume | Final Concentration |
10X Pfu Buffer (with Mg2+) | 5µl | 1 X |
dNTP mix, 10mM each | 1µl | 200µM each |
upstream primer | 5–50pmol | 0.1–1.0µM |
downstream primer | 5–50pmol | 0.1–1.0µM |
DNA template | variable | <0.5µg/50µl |
Pfu DNA Polymerase (5U/µl) | variable | 1.25U/50µl |
DEPC-treated Water | Up to 50µl | - |
2. Recommended thermal cycling conditions for Pfu DNA Polymerase-mediated PCR amplification as follows:
*The annealing temperature for a specific amplification reaction will depend upon the sequences of the two primers.
Note: 1. It is critical to withhold Pfu DNA Polymerase until after the addition of dNTPs; otherwise, the proofreading activity of the polymerase may degrade the primers, resulting in nonspecific amplification and reduced product yield.
2. Assemble on ice.
In the experiment, the 773bp target fragment was amplified by PCR using PUC57 as the template. At the same time, competing enzymes were added to investigate the enzyme activity of Pfu DNA Polymerase. As shown in the figure, this product has the following effects.
Lane 1 Negative Control (negative control with no added enzyme only);
Lane 2 UA-Pfu DNA Polymerase 2.5U;
Lane 3 UA-Pfu DNA Polymerase 5U;
Lane 4 Competing product A 5U;
Lane 5 Competing product B 5U;
1μg (R: reducing condition, N: non-reducing condition).
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