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:
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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.
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Ultrapure, recombinant proteins are used to prepare Perpetual OptiTaq DNA Polymerase.
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Our carefully selected anti-Taq antibodies have high thermal stability, providing protection against non-specific primer extension from room temperature to 70°C.
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The polymerase activity is restored during the initial denaturation step when amplification reactions are heated at 92-95°C for two minutes.
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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.
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High stability of the complexes allows for the enormous increase of PCR specificity, sensitivity and yield in comparison to the conventional PCR assembly method.
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Automatic "hot start" PCR is a convenient method when assembling multiple PCR reactions, saving time and reagents.
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Clean and safe laboratory practice assured, due to removed necessity to open hot tubes.
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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).
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Enables increased amplification product yield in comparison with Taq DNA polymerase over wide range of PCR products.
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Maintains the 5'´->3´ exonuclease activity.
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Adds extra A at the 3' ends.
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Suitable for multiplex PCR due to increased specificity, wider tolerance for Mg2+, salts concentration, and pH (2,3).
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Improves PCR results with critical templates, such as containing GC-rich regions, palindromes or multiple repeats.
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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:
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Cline, J., Braham, J. and Hogrefe, H. (1996) Nucleic Acids Res. 24, 3545.
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Chien, A., Edgar, D.B. and Trela, J.M. (1976) J. Bacteriol. 127, 1550.
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Kaledin, A.S., Sliusarenko, A.G. and Gorodetskii, S.I. (1980) Biokhimiya 45, 644.