Mixture of stable thermophilic DNA polymerases capable of generating PCR products up to 20 kb with high fidelity;, suitable for applications requiring high temperature synthesis of DNA.
The enzyme is supplemented with two inert gel tracking dyes.
Description:
-
OptiTaq DNA Polymerase is a modified and optimized thermostable enzymes blend containingThermus aquaticus DNA polymerase and Pyrococcus sp. DNA polymerase.
-
Ultrapure, recombinant enzymes are used to prepare OptiTaq DNA Polymerase.
-
The enzymes blend exhibits the 3´->5´ proofreading activity, resulting in considerably higher PCR fidelity and processivity 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 as it exhibits 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.
-
Color 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.
-
Use of Color OptiTaq DNA Polymerase offers several advantages:
-
visualizes the addition of the polymerase to the reaction,
-
confirms complete mixing,
-
enables direct loading of PCR products onto an agarose gel without addition of a gel loading buffer,
-
the added dyes allow to track electrophoresis progress,
-
do not affect PCR performance,,
-
do not interfere with most downstream applications (exeption: the polymerase is not recommended for any downstream application using absorbance or fluorescence excitation).
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.
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:
-
Cline, J., Braham, J. and Hogrefe, H. (1996) Nucleic Acids Res. 24, 3545.
-
Chien, A., Edgar, D.B. and Trela, J.M. (1976) J. Bacteriol. 127, 1550.
-
A.S.,Sliusarenko, A.G. and Gorodetskii, S.I. (1980) Biokhimiya 45, 644.