Extremely thermostable proofreading DNA polymerase blend, formulated for efficient site-directed mutagenesis and synthesis of DNA products up to 20 kb.
Unit Definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmoles of dNTP into acid-insoluble material in 30 min at 74°C. The reaction conditions are: 50 mM Tris-HCl (pH 9.0 at 25oC), 50 mM NaCl, 5 mM MgCl2, 200 μM each of dATP, dCTP, dGTP, dTTP (a mix of unlabeled and [3H]dTTP), 10 μg activated calf thymus DNA and 0.1 mg/ml BSA in a final volume of 50 μl.
Storage Conditions: Store at -20°C or at –70oC.
Description:
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PfuPlus! is a modified and optimized hyperthermophilic Pfu DNA Polymerase (1) blended with thermostable polymerisation enhancing factor.
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Ultrapure recombinant enzyme.
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The enzyme catalyzes the polymerization of nucleotides into duplex DNA in the 5'->3' direction in the presence of magnesium ions.
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The enzyme exhibits the 3'->5' proofreading activity, resulting in over 10-fold higher PCR fidelity than possible with Taq DNA Polymerases (2).
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The constituent of PfuPlus! DNA Polymerase, the polymerase-enhancing factor, enhances PCR product yields and increases target length capability of Pfu DNA Polymerase.
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Enhanced performance of PfuPlus! DNA Polymerase allows to use fewer PCR cycles and lower DNA template concentrations when compared to Pfu DNA Polymerase.
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PfuPlus! DNA Polymerase is recommended for use in high-fidelity PCR, PCR of GC-rich sequences or problematic secondary structures, site-directed mutagenesis and cloning of blunt-ended PCR products.
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The enzyme is also recommended for general use in PCR and primer extension reactions at elevated temperatures to obtain a wide range of PCR products up to 20 kb.
Storage Buffer: 50 mM Tris-HCl (pH 8.0 at 22oC), 0.1 mM EDTA, 1 mM dithiothreitol, 50% glycerol and stabilizers.
10 x Reaction Buffer:
10 x Pfu Buffer: The buffer contains 20 mM MgSO4.
Quality Control: All preparations are assayed for contaminating 3'-exonuclease, and nonspecific single- and double-stranded DNase activities. Typical preparations are greater than 95% pure, as judged by SDS polyacrylamide gel electrophoresis.
References:
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Lundberg, K., Shoemaker, D., Adams, M., Short, J., Sorge, J. and Mathur E. (1991) Gene 108, 1.
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Cline, J., Braham, J. and Hogrefe, H. (1996) Nucleic Acids Res. 24, 3546.
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Kaledin, A.S., Sliusarenko, A.G. and Gorodetskii, S.I. (1980) Biokhimiya 45, 644.