T7 RNA polymerase is highly specific for the bacteriophage T7 promoter and terminator sequences. It is extensively used to prepare RNA transcripts for stuctural and metabolic studies. The RNA transcripts can be converted to probes for sensitive hybridization detection studies. T7 polymerase and dideoxynucleotides can be used to directly sequence DNA.
Components
T7 RNA Polymerase is supplied as a solution of 100 mM NaCl, 50 mM Tris-HCl (pH 7.9), 0.1 mM EDTA, 0.1% Triton X-100, 1 mM DTT, and 50% (v/v) glycerol.
Unit Definition
One unit will catalyze the incorporation of 1 nmol of rNTP into acid-precipitable material in 60 min at 37°C.
Analysis Note
Activity assay: 40 mM Tris-HCl, pH 7.9, 6 mM MgCl2, 4 mM spermidine, 10 mM DTT, 0.5 μM each rNTP + 10 μCi α-32P-UTP, 3-10 units of enzyme, and 1 μg of a 350 bp template are incubated for 10 min at 37°C in a total volume of 100 μl. Typical results are ≥50% incorporation of labeled nucleotide into ≥90% full-length transcript.
Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme.
M Chamberlin et al.
The Journal of biological chemistry, 248(6), 2235-2244 (1973-03-25)
Science (New York, N.Y.), 339(6121), 816-819 (2013-02-16)
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