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DESCRIPTION
T7 RNA Polymerase is a
DNA-dependent RNA polymerase that recognizes and initiates synthesis of
RNA on double-stranded DNA templates that carry the T7 phage promoter. The
polymerase is used in vitro to generate large quantities of RNA
complementary to one strand of foreign DNA that has been cloned
immediately downstream from the promoter in plasmids specifically designed
for this purpose.
SOURCE
Recombinant strain of E. coli.
APPLICATIONS
¡Ü Preparation of RNA probe
for hybridization.
¡Ü Generation of RNA substrates for studies of RNA
processing.
¡Ü Generation of RNA for in vitro translation.
UNIT DEFINITION
One unit is defined as
the amount of enzyme required to catalyze the incorporation of 1nmole of
nucleotide triphosphate into acid insoluble product in 60 minutes at 37¡É.
STANDARD UNIT ASSAY CONDITIONS
40mM
Tris-HCl (pH7.9), 6mM MgCl2, 10mM DTT, 2mM spemidine, 0.5mM ATP, GTP, CTP,
and UTP, 0.5¥ìCi [3H-CTP], and 1¥ìg T7 promoter-containing plasmid.
CONCENTRATION: 20-50 units/¥ìl.
10X REACTION BUFFER
400mM Tris-HCl
(pH7.9), 60mM MgCl2, 20mM spermidine, 100mM DTT.
STORAGE CONDITIONS
50mM Tris-HCl
(pH7.9), 100mM NaCl, 1mM EDTA, 20mM 2-mercaptoethanol, 0.1% Triton X-100,
and 50% glycerol. Store at -20¡É.
REFERENCES
1. Krieg, P. A. and Melton,
D. A. (1987) Methods Enzymol. 155, 397.
2. Davanloo, P., et al. (1984)
Proc. Natl. Acad. Sci. USA 81, 2035.
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