Perpetual OptiTaq DNA Polymerase (Hot Start OptiTaq DNA Polymerases)

Monoclonal antibody automatic "hot start" proofreading Taq/Pfu PCR system

Mixture of thermostable Taq DNA Polymerase, proofreading Pfu DNA Polymerase, anti-Taq DNA Polymerase antibodies, reversible inhibitors and enhancers for automatic "hot start" PCR. The blend generates products up to 20 kb with stringent amplification specificity, sensitivity, fidelity and yield.

Description:

• Perpetual OptiTaq DNA Polymerase is a modified and balanced blend containing top qualityThermus aquaticus DNA Polymerase, Pyrococcus furiosus DNA Polymerase, anti-Taq DNA Polymerase antibodies, reversible inhibitors and enhancers.
• Ultrapure, recombinant proteins are used to prepare Perpetual OptiTaq DNA Polymerase.
• Our carefully selected anti-Taq antibodies have high thermal stability, providing protection against non-specific primer extension from room temperature to 70°C.
• The polymerase activity is restored during the initial denaturation step when amplification reactions are heated at 92-95°C for two minutes.
• Formation of inactive complexes between Taq DNA Polymerase and an anti-Taq antibody forms a basis for automatic "hot start" PCR, which allows for the assembly of PCR reactions at room temperature.
• High stability of the complexes allows for the enormous increase of PCR specificity, sensitivity and yield in comparison to the conventional PCR assembly method.
• Automatic "hot start" PCR is a convenient method when assembling multiple PCR reactions, saving time and reagents.
• Clean and safe laboratory practice assured, due to removed necessity to open hot tubes.
• The blend catalyzes the polymerization of nucleotides into duplex DNA in the 5'->3' direction in the presence of magnesium ions and exhibits the 3'->5' proofreading activity, resulting in considerably higher PCR fidelity than possible with unmodified Taq DNA polymerase (1).
• Enables increased amplification product yield in comparison with Taq DNA polymerase over wide range of PCR products.
• Maintains the 5'´->3´ exonuclease activity.
• Adds extra A at the 3' ends.
• Suitable for multiplex PCR due to increased specificity, wider tolerance for Mg²⁺, salts concentration, and pH (2,3).
• Improves PCR results with critical templates, such as containing GC-rich regions, palindromes or multiple repeats.
• Perpetual OptiTaq DNA Polymerase is recommended for use in PCR and primer extension reactions at elevated temperatures to obtain a wide range 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 minutes at 74°C. The reaction conditions are: 50 mM Tris-HCl (pH 9.0 at 25°C), 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 Buffer: 20 mM Tris-HCl (pH 8.0 at 22°C), 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 50% glycerol and stabilizers.

Storage Conditions: Store at –20°C.

10 x Reaction Buffer:

10 x Pol Buffer A (optimization buffer without MgCl₂): the buffer allows to optimize MgCl₂concentration.

10 x Pol Buffer B (general application, up to 20 kb): the buffer contains 15 mM MgCl₂ and is optimized for use with 0.2 mM of each dNTP..

10 x Pol Buffer C (coloured): 10 x Pol Buffer B enriched with two gel tracking dyes and a gel loading reagent. The buffer enables direct loading of PCR products onto an agarose gel.

Quality Control: All preparations are assayed for contaminating endonuclease, 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. Cline, J., Braham, J. and Hogrefe, H. (1996) Nucleic Acids Res. 24, 3545.
2. Chien, A., Edgar, D.B. and Trela, J.M. (1976) J. Bacteriol. 127, 1550.
3. Kaledin, A.S., Sliusarenko, A.G. and Gorodetskii, S.I. (1980) Biokhimiya 45, 644.