T7 RNA Polymerase is a DNA-dependent RNA polymerase from T7 phage ， that possesses a strong and specific 5 ´→ 3 ´ RNA polymerase activity. T7 RNA Polymerase has high specificity for T7 promoter sequences and will synthesize large quantities of RNA from a DNA fragment inserted downstream from a promoter.
Transportation and Storage
Transportation under 0℃ and storage at -20℃; Validity: 24 months.
Unit Definition
One unit is defined as the amount of enzyme required to incorporate 1 nmol ATP into an acid-insoluble material in 1 hour at 37°C.
Quality Control Assays
- Endonuclease Activity: Incubation of a 40 μL reaction containing a minimum of 200 U of T7 RNA Polymerase with 4 μg pUC19 DNA for 16 hours at 37℃ results in no detectable degradation as determined.
- Exonuclease Activity: Incubation of a 50 µL reaction containing a minimum of 200 U of T7 RNA Polymerase with 1 μg Hind Ⅲ digest λ DNA for 16 hours at 37℃ results in no detectable degradation as determined.
- Nickase Activity: Incubation of a 50 µL reaction containing a minimum of 200 U of T7 RNA Polymerase with 1 μg pBR322 DNA for 16 hours at 37℃ results in no detectable degradation as determined.
- RNase Activity: Incubation of a 50 µL reaction containing a minimum of 200 U of T7 RNA Polymerase with 1.6 μg MS2 RNA for 4 hours at 37℃ results in no detectable degradation as determined.
- Heat Inactivation: 75℃ for 10min.
RNA Synthesis Reaction
| Reagent | Amount |
|---|---|
| Nuclease-free H2O | Up to 20 μL |
| 10 × HY T7 Buffer | 2 μL |
| ATP/CTP/GTP/UTP (100 mM each) | 2 μL each (10 mM each Final) |
| RNase Inhibitor (40 U/μL) | 1 μL |
| Pyrophosphatase Inorganic (0.1 U/μL) | 2 μL |
| High Yield T7 Polymerase (<2000 nt) | 2 μL |
| Linearized DNA Template | 1 μg |
Incubation Time: 37℃ for 1-2 hours.
Stop of Reaction: Add 2 µL 0.2 M EDTA (pH=8.0@25℃) or heat to 75°C for 10min.
DNA Removal: DNA template can be removed with 2U DNase I (RNase-free) and incubation for 15 min at 37°C.
Inhibitors: Metal chelators, enzyme activity is reduced by 50% at NaCl or KCl concentration above 150 mM.
Precaution
- This product is suitable for in vitro transcription of RNA with length less than 2000
- The transcription reaction should be performed under contamination without RNases. Wearing gloves is advisable. The tips, tubes and water should be nuclease free. All the solutions should be made up in nuclease free water.
- The RNA synthesis reaction mixture should be prepared at room temperature, since DNA may precipitate in the presence of spermidine at 4°C.
- The yield of proper length transcripts decreases if the template DNA is incompletely linearized.
- The reaction mixture can be scaled up or down.
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