What is T7 RNA Polymerase?
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.
T7 RNA Polymerase
Transportation and Storage
Transportation under 0℃ and storage at -20℃; Validity: 24 months.
|T7 RNA Polymerase (50 U/μL)||0.1 mL||1 mL|
|10×HH T7 Buffer||0.1 mL||1 mL|
|T7 RNAPolymerase (50 U/μL)||10 mL||100 mL|
|10×HH T7 Buffer||10 mL||100 mL|
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 HindⅢ 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
|Nuclease-free water||Up to 20 μL|
|10×HH T7 Buffer||2 μL|
|ATP/GTP/CTP/UTP (10 mM each)||2 μL each (1 mM each Final)|
|RNase Inhibitor (40 U/μL)||1 μL|
|Pyrophosphatase Inorganic (0.1 U/μL)||2 μL|
|T7 RNA Polymerase (50 U/μL)||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.
- 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.
Excellent RNA output rate; homogeneous transcript length;transcript length up to 10K.
Application of T7 RNA Polymerase
Radiolabeled RNA probes
Non-isotopic RNA labeling
Preparation of RNA vaccines
Guide RNA for gene targeting
mRNA for in vitro translation and micro injection
RNA structure, processing and catalysis studies
Anti-sense RNA for gene expression experiment