The experiment was designed. 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 541nt. Then 5μl reaction product was removed, 1μl 6X 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.
Marker (100~2000 bp)
Lane 1 Negative Control (negative control with no added enzyme only);
Lane 2 50U UA-T7 RNAP;
Lane 3 50U Competitor N-T7 RNAP;
T7 RNA Polymerase
T7 RNA Polymerase
Product Details
Product Details
Product Specification
| Synonyms | T7 RNAPol、T7 RNAP |
| Expression System | E.coli |
| Molecular Weight | 100 kDa (Reducing) |
| Purity | ＞95% by SDS-PAGE |
| Physical Appearance | Liquid |
| Storage Buffer | 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 |
| Stability & Storage | Store at -25 ~ -15℃ for 2 years |
| Reference | 1. Lawyer F C,Stoffel S,Saiki R K ,et al. Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus.[J].Journal of Biological Chemistry, 1989, 264. |
Background
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.
Components
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)
Protocol
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 Polymerase |
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.
Guidelines
Please avoid repeated freeze-thaw cycles.
Unit Definition
Picture
Picture
Bioactivity
SDS-PAGE
2μg (R: reducing condition, N: non-reducing condition).
SEC-HPLC
95.2%
