T7 RNA polymerase

Product Information

Product description: T7 RNA Polymerase is a monomeric bacteriophage encoded DNA directed RNA polymerase which catalyzes the formation of RNA in the 5'-3' direction. In the process of initiation of transcription T7 recognizes a specific promoter sequence, the T7 promoter. T7 consists of 883 amino acids and has a molecular weight of 99kDa.

Source: Recombinant E.coli

Concentration: 50/200/1000U/μL (KU/mL)

Unit Definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 1 nmol of rATP into acid-insoluble product in 1 hour at 37℃.

Storage Buffer: 20mM Tris-HCl PH 7.9, 100mM NaCl, 10mM DTT, 0.1% Triton X-100, 1mM EDTA, 50%(v/v) Glycerol.

Companion Product: 10X IVT Reaction Buffer, Cat#ON-62, 400mM Tris PH7.9, 100mM DTT, 20mM Spermidine, 60mM MgCl₂.
10X IVT Reaction Buffer without Mg²⁺, Cat#ON-41, 400mM Tris PH7.9, 100mM DTT, 20mM Spermidine.

Key points of operation

1. T7 RNA Polymerase is extremely sensitive to salt inhibition. For best overall salt concentration should not exceed 50mM.

2. Higher yields of RNA may be obtained by raising NTP concentrations (up to 10mM each).

Mg²⁺ concentration should be raised to 4~10mM above the total NTP concentration.

T7 RNA Polymerase should be raised to 30K units/mL.

Additionally, inorganic pyrophosphatase should be added to a final concentration of 4 units/mL.

3. 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 20mM fresh dithiothreitol.

Or make a reaction buffer without DTT and add fresh DTT when preparing reaction mixture.

Security Information

Storage Conditions: -20℃

Quality Assurance: Free of endonuclease, exonuclease and RNase activities.

Physical Purity: ＞95% by SDS-PAGE.

Reference

1. Chamberlin, M., McGrath, J. & Waskell, L. Nature 228, 227–31 (1970).

2. Davanloo, P., Rosenberg, A. H., Dunn, J. J. & Studier, F. W. Proc. Natl. Acad. Sci. U. S. A. 81, 2035–9 (1984).