SP6 RNA Polymerase is a
DNA-dependent RNA polymerase that recognizes and initiates synthesis of
RNA on double-stranded DNA templates that carry the SP6 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.
Recombinant strain of E. coli.
¡Ü Preparation of RNA probe
¡Ü Generation of RNA substrates for studies of RNA
¡Ü Generation of RNA for in vitro translation.
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
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 SP6 promoter-containing plasmid.
CONCENTRATION: 10-20 units/¥ìl.
10X REACTION BUFFER
(pH7.9), 60mM MgCl2, 20mM spermidine, 100mM DTT.
phosphate (pH7.7), 100mM NaCl, 1mM EDTA, 10mM DTT, 0.1% Triton X-100, and
50% glycerol. Store at -20¡É.
1. Krieg, P. A. and Melton,
D. A. (1987) Methods Enzymol. 155, 397.
2. Butler, E. T. and
Chamberlain M. J. (1982) J. Biol. Chem. 257, 5772.