Description:
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Stable thermophilic DNA polymerase (1).
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Synthesizes cDNA from RNA template in high temperature.
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Results in greater specificity of primer hybridization and extension.
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Minimizes problems with strong secondary structure of RNA.
Unit Definition: One unit is the amount of enzyme required to incorporate 10 nmoles of total deoxynucleotides into acid-insoluble material in 30 min at 70°C.
Storage Conditions: Store at –20°C.
Storage Buffer: 50 mM Tris-HCl (pH 7.5 at 22°C), 5 mM dithiothreitol, 0.1 mM EDTA, 50% (v/v) 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 10 kb): the buffer contains 15 mM MgCl2 and is optimized for use with 0.2 mM of each dNTP.
Assay Conditions: 25 mM Tris-HCl (pH 9.5 at 25°C), 10 mM MgCl2, 1 mg/ml of bovine serum albumin, 50 mM KCl, 0.2 mM each dCTP, dGTP, dTTP, and [a-³²P]dATP and 15 µg of activated DNA. Incubation is at 70oC for 10 min in a reaction volume of 50 µl.
Quality Control: All preparations are assayed for contaminating endonuclease, 3'-exonuclease, and nonspecific single- and double-stranded DNase activities. Typical preparations are greater than 90% pure, as judged by SDS polyacrylamide gel electrophoresis.
References:
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Wang, A.M., Doyle, M.V., and Mark, D.F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9717-9721.