100 kDa (Reducing)
Store at -25 ~ -15℃ for 2 years
[1] Athanasios D , Kanchana R ,Hobert Elissa M.Moore Melissa J.Rabideau Amy E.An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts[J].Nature biotechnology, 2023, 41(4):560-568.
[2]Skinner, and M. G. . "Promoter Binding, Initiation, and Elongation By Bacteriophage T7 RNA Polymerase A SINGLE-MOLECULE VIEW OF THE TRANSCRIPTION CYCLE." Journal of Biological Chemistry 279.5(2004):3239-44.
Bacteriophage T7 RNA Polymerase is a DNA-dependent RNA polymerase that is highly specific for the T7 phage promoters. The 99 KD enzyme catalyzes in vitro RNA synthesis from a cloned DNA sequence under the T7 promoters. RNA produced using the T7 RNA Polymerase is suitable for many applications in research and biotechnology.
Storage Solution: 50 U/ul T7 RNA Polymerase, 50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 10 mM DTT, 50%(v/v) Glycerol, 0.1% (w/v) Triton® X-100 pH 7.9 @ 25°C
10* Reaction Buffer: 400 mM Tris-HCl, 60 mM MgCl2, 10 mM DTT, 20 mM spermidine (pH 7.9 @ 25°C)
1. Assemble the reaction at room temperature in the following order.
Components | Volume | Final Concentration |
10X Reaction Buffer | 2 µl | 1 X |
NTP mix, 10 mM each | 1 µl | 0.5 mM each |
RNase Inhibitor（40 U/ul） | 0.5 µl | 1 U/µl |
50 mM DTT | 2 µl | 5 mM |
Template DNA | variable | 20 ng–1 µg |
T7 RNA Pol | 1 µl | 2.5 U/µl |
DEPC-treated Water | Up to 20µl | - |
2. Incubate at 37°C for 1 hour. For shorter (< 300 nt) transcripts incubate at 37°C for 2–16 hours.
Please avoid repeated freeze-thaw cycles.
The experimental design was carried out as follows. In a 20µl reaction system (40mM Tris-HCl pH7.9, 2mM Spermidine, 6mM MgCl2, 1mM DTT), PCR products containing T7 Promoter were added as template DNA and incubated at 37℃ for 1h. The reaction was terminated after incubation at 70℃ for 10min, and 10U DNase I was added to digest the template DNA. The RNA transcription product obtained was 178nt. Then 9μl reaction product was removed, 1μl 10X Loading Buffer was added, and 1% agar-gel was used for electrophoresis at room temperature, 1X TAE was used as the electrophoresis solution, 160V for 20min. After electrophoresis, the results were photographed. As shown in the figure, this product has excellent transcription effect.
Lane 1 Negative Control (negative control with no added enzyme only);
Lane 2 UA-T7 RNA Polymerase 3.125U;
Lane 3 UA-T7 RNA Polymerase 6.25U;
Lane 4 UA-T7 RNA Polymerase 12.5U;
Lane 5 UA-T7 RNA Polymerase 25U;
Lane 6 UA-T7 RNA Polymerase 50U;
1μg (R: reducing condition, N: non-reducing condition).
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