T7 RNA Polymerase
Bacteriophage T7 RNA Polymerase is a DNA-dependent RNA polymerase with high specificity for the T7 promoter. This enzyme catalyzes the 5’→3’synthesis of RNA from DNA downstream from its promoter.
T7 RNA Polymerase, T7 RNA聚合酶
Source:
Escherichia coli
Purity:
>98% as determined by SDS-PAGE. Purified by Ni-NTA chromatography.
Unit Definition:
One unit is defined as the amount of the enzyme incorporates 1 nmol of ATP into acid-insoluble product in 1 hour at 37°C.
Reaction Condition:
1X RNA Polymerase Reaction Buffer, supplemented with 0.5 mM each ATP, UTP, GTP, CTP, and DNA template containing the T7 RNA Polymerase promoter. Incubate at 37°C.
10X RNA Polymerase Reaction Buffer: 400 mM Tris-HCl (pH 8.0), 60 mM MgCl2, and 20 mM spermidine.
Storage Buffer:
T7 RNA Polymerase is supplied in 100 mM Tris-HCl (pH 7.9), 20 mM KCl, 1 mM DTT, 1 mM EDTA, 0.1% Triton® X-100 and 50% (v/v) glycerol.
Storage:
Stored at -20°C. For optimal storage, aliquot the enzyme, reaction buffer and DTT reagent into smaller quantities and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles.
Notes:
1. Transcription reaction should be performed under RNase free condition. Use nuclease-free tubes, reagents, and water to avoid RNase contamination. Also, wear gloves when working with RNA.
2. To obtain optimal condition, NTP concentration can be titrated between 5 – 10 mM.
3. The volume of T7 RNA Polymerase can be titrated between 1-2 μL in the IVT reaction to optimize your assay.
Escherichia coli
Purity:
>98% as determined by SDS-PAGE. Purified by Ni-NTA chromatography.
Unit Definition:
One unit is defined as the amount of the enzyme incorporates 1 nmol of ATP into acid-insoluble product in 1 hour at 37°C.
Reaction Condition:
1X RNA Polymerase Reaction Buffer, supplemented with 0.5 mM each ATP, UTP, GTP, CTP, and DNA template containing the T7 RNA Polymerase promoter. Incubate at 37°C.
10X RNA Polymerase Reaction Buffer: 400 mM Tris-HCl (pH 8.0), 60 mM MgCl2, and 20 mM spermidine.
Storage Buffer:
T7 RNA Polymerase is supplied in 100 mM Tris-HCl (pH 7.9), 20 mM KCl, 1 mM DTT, 1 mM EDTA, 0.1% Triton® X-100 and 50% (v/v) glycerol.
Storage:
Stored at -20°C. For optimal storage, aliquot the enzyme, reaction buffer and DTT reagent into smaller quantities and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles.
Notes:
1. Transcription reaction should be performed under RNase free condition. Use nuclease-free tubes, reagents, and water to avoid RNase contamination. Also, wear gloves when working with RNA.
2. To obtain optimal condition, NTP concentration can be titrated between 5 – 10 mM.
3. The volume of T7 RNA Polymerase can be titrated between 1-2 μL in the IVT reaction to optimize your assay.
Standard RNA synthesis procedures:
1. Below reaction mixture should be prepared under room temperature and combined in the following order:
2. Incubate at 37°C for 30 minutes to 2 hours.
3. Above reaction mixture may be scaled up or down proportionately.
1. Below reaction mixture should be prepared under room temperature and combined in the following order:
| Component | Amount | Final concentration |
| Nuclease-Free H2O | X μL | - |
| Template DNA | 0.5-1 μg | - |
| 10X RNA Polymerase Reaction Buffer | 2 μL | 1X |
| ATP (100 mM) | 1 μL | 5 mM |
| UTP (100 mM) | 1 μL | 5 mM |
| CTP (100 mM) | 1 μL | 5 mM |
| GTP (100 mM) | 1 μL | 5 mM |
| 100 mM DTT | 2 μL | 10 mM |
| T7 RNA Polymerase (200 U/μL) | 1 μL | - |
| RNase inhibitor (optional) | 0.5 μL | 1U/μL |
| Total reaction volume | 20 μL | - |
3. Above reaction mixture may be scaled up or down proportionately.