Mixture of stable thermophilic DNA polymerases and enhancing factors, capable of generating over 20 kb products with high fidelity; suitable for applications requiring high temperature synthesis of DNA.
Description:
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OptiTaq DNA Polymerase is a modified and optimized thermostable enzymes blend containingThermus aquaticus DNA polymerase, Pyrococcus furiosus DNA polymerase and enhancing factors.
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Ultrapure, recombinant enzymes are used to prepare OptiTaq DNA Polymerase.
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The enzymes blend 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 as it exhibits 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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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 min 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 Conditions: Store at –20°C.
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.
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, 3546.
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Chien, A., Edgar, D.B. i Trela, J.M. (1976) J. Bacteriol. 127, 1550.
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Kaledin, A.S., Sliusarenko, A.G. i Gorodetskii, S.I. (1980) Biokhimiya 45, 644.