MCLAB :: Products :: Enzymes :: T7 RNA Polymerase

T7 RNA Polymerase

Catalog No.	Size	Price
RP-200	5,000 U	$58.00
RP-210	20,000 U	$185.00
RP-OEM	Please inquire

Ideal for RNA synthesis. For Research Use Only.

Overview
T7 RNA Polymerase is a DNA-dependent RNA polymerase that catalyzes the formation of RNA in the 5'? 3' direction. It is extremely promoter-specific and only transcribes bacteriophage T7 DNA or DNA cloned downstream of a T7 promoter. Its source is the T7 bacteriophage, which is a virus that infects only bacteria.

Applications

Radiolabeled RNA probe preparation
RNA generation for in vitro translation
RNA generation for studies of RNA structure, processing and catalysis
Expression control via anti-sense RNA

Advantages

Has extremely high specificity for promoter sequences found in T7 bacteriophageDNA and in various cloning vectors containing the T7 promoter (eg. pCR®II-TOPO®, pCR®-BluntII-TOPO®, pET-DEST42)

Recommended reaction conditions

1X RNAPol Reaction Buffer: 40 mM Tris-HCl, 6 mM MgCl2, 10 mM Dithiothreitol, 2 mM spermidine, pH 7.9 @ 25°C
Supplemented with 0.5 mM ATP, 0.5 mM UTP, 0.5 mM Guanosine triphosphate, and 0.5 mM CTP
Incubate at 37°C.

Recommended reaction conditions

1X RNA Polymerase Reaction Buffer: 40 mM Tris-HCl, 6 mM MgCl2, 10 mM Dithiothreitol, 2 mM spermidine, pH 7.9 @ 25°C
Supplemented with 0.5 mM ATP, 0.5 mM UTP, 0.5 mM Guanosine triphosphate, and 0.5 mM CTP
Incubate at 37°C.

Unit Definition
One unit is defined as the amount of enzyme that will incorporate 1 nmol ATP into acid-insoluble material in a total reaction volume of 50 µl in 1 hour at 37°C in 1X RNA Polymerase Reaction Buffer.

Concentration
50,000 units/ml

Recommended storage conditions

Storage buffer: 50 mM Tris-HCl, 100 mM NaCl, 20 mM 2-Mercaptoethanol, 1 mM EDTA, 50% Glycerol, 0.1% Triton X-100, pH 7.9 @ 25°C
Storage temperature: -20°C

Notes

Dithiothreitol is required for activity.
T7 RNA Polymerase is extremely sensitive to salt inhibition.
For best overall salt concentration should not exceed 50 mM.
Higher yields of RNA may be obtained by raising NTP concentrations (up to 4 mM each). Mg2+ concentration should be raised to 4 mM above the total NTP concentration. Additionally, inorganic pyrophosphatase should be added to a final concentration of 4 units/ml.
An apparent decrease in enzyme activity over time may be due to the breakdown of dithiothreitol in the reaction buffer; even when stored at -20°C. If you observe a decrease in yield, try supplementing your reactions with a final concentration of 10 mM fresh dithiothreitol.

References

Schenborn, E.T. and Meirendorf, R.C. (1985) Nucl. Acids Res., 13, 6223-6236.
Davanloo, P., Rosenberg, A.H., Dunn, J.J. and Studier, F.W. (1984) Proc. Natl. Acad. Sci. USA, 81, 2035-2039.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd Ed.), 10.27-10.37.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd Ed.), 18.82-18.84.
Melton, D.A., Kreig, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984) Nucl. Acids Res., 12, 7035-7056.
Milligan, J.F., Groebe, D.R., Witherell, G.W. and Uhlenbeck, O.C. (1987) Nucl. Acids Res., 15, 8783.
Noren, C.J. et al. (1990) Nucl. Acids Res., 18, 83-88.
Kreig, P.A. and Melton, D.A. (1984) Nucl. Acids Res., 12, 7057-7070.