T3
RNA Polymerase
T3 RNA Polymerase is a highly processive enzyme that shows strict specificity for its double stranded promoter sequence (5´ AATTAACCCTCACTAAAG 3´) and catalyzes the 5’ - 3’ synthesis of RNA.
The T3 RNAP has been obtained from E. coli BL21 cells with a cloned gene encoding the enzyme and purified using our ArtPure™ proprietary purification process, yielding a >99% pure, animal origin-free product that is free of contaminants such as proteinases, RNases and DNases and has no added enzyme inhibitors.
Suggested IVT Protocol
Component | Amount |
|---|---|
Linearized DNA template | 500 ng |
Transcription buffer (10X) | 1 µl |
NTP | 1 mM each |
RNase inhibitor | 1 U/µl |
T7 RNA Polymerase | 1 µl |
Nuclease-free water | to 10 µl |
Incubate the reaction 1 hour at 37°C.
To remove the DNA template, add 1 U of DNase I and incubate at 37°C for 15 minutes.
Inactivate the DNase I by adding 5 mM of EDTA, incubate at 70°C for 10 minutes.
Enzyme Quality
Purity
Activity
RNases
Proteinases
Nucleases
Single-stranded exonucleases
Double-stranded endonucleases
Double-stranded exonucleases
0h 4h 16h 0h 4h 16h 0h 4h 16h
Definitions
Activity (U/µl): One enzyme unit incorporates 1 nmol of soluble RNA nucleotides into insoluble polymeric RNA per hour at 37°C.
Purity: Evaluated by SDS-PAGE, where the integrated peak area of the RNA Polymerase is compared to other protein peaks in the same lane.
Transcriptional activity: Quantification of total RNA products after in vitro transcription and RNA purification.
Proteinases: Assessed using Pierce™ Fluorescent Protease Assay Kit (ThermoFisher Scientific) following the manufacturer’s guidelines.
RNases: Assessed using the RNAse Alert kit (Integrated DNA Technologies), following the manufacturer’s guidelines.
Double-stranded exonucleases: 2.5 µl of the final enzyme product are incubated with 0.5 µg of linearized double-stranded DNA substrate (DNA ladder) for 0 hours, 4 hours at 37°C and 16 hours at room temperature.
Double-stranded endonucleases: 2.5 µl of the final enzyme product are incubated with 0.5 µg of supercoiled plasmid DNA for 0 hours, 4 hours at 37°C and 16 hours at room temperature.
Single-stranded exonucleases: 2.5 µl of the final enzyme product are incubated with 0.5 µg of ssDNA substrate (calf-thymus DNA) for 0 hours, 4 hours at 37°C and 16 hours at room temperature.
Product Information
Storage upon receipt: Our T3 RNA Polymerase is stable at -20°C for up to 12 months in the glycerol-based supplied buffer. For extended storage, we recommend aliquoting in smaller volumes, freezing at -80°C and avoiding repeated freeze-thaw cycles.
Storage buffer: 50 mM Tris-HCl, 100 mM NaCl, 20 mM DTT, 1 mM EDTA, 50% glycerol, 0.1% Triton X-100, pH 7.9.
Transcription buffer (10X): 400 mM Tris-HCl, 60 mM MgCl2, 100 mM DTT, 100 mM NaCl, 20 mM spermidine, pH 7.9.