PfuPlus! DNA Polymerase (Pyrococcus sp.) (High Fidelity DNA Polymerase)

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 25^oC), 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 –70^oC.

Description:

• PfuPlus! is a modified and optimized hyperthermophilic Pfu DNA Polymerase (1) blended with thermostable polymerisation enhancing factor.
• Ultrapure recombinant enzyme.
• The enzyme catalyzes the polymerization of nucleotides into duplex DNA in the 5'->3' direction in the presence of magnesium ions.
• The enzyme exhibits the 3'->5' proofreading activity, resulting in over 10-fold higher PCR fidelity than possible with Taq DNA Polymerases (2).
• The constituent of PfuPlus! DNA Polymerase, the polymerase-enhancing factor, enhances PCR product yields and increases target length capability of Pfu DNA Polymerase.
• Enhanced performance of PfuPlus! DNA Polymerase allows to use fewer PCR cycles and lower DNA template concentrations when compared to Pfu DNA Polymerase.
• 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.
• 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 22^oC), 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:

1. Lundberg, K., Shoemaker, D., Adams, M., Short, J., Sorge, J. and Mathur E. (1991) Gene 108, 1.
2. Cline, J., Braham, J. and Hogrefe, H. (1996) Nucleic Acids Res. 24, 3546.
3. Kaledin, A.S., Sliusarenko, A.G. and Gorodetskii, S.I. (1980) Biokhimiya 45, 644.