SP6
RNA Polymerase
SP6 RNA Polymerase is a highly processive enzyme that shows strict specificity for its double stranded promoter sequence (5´ ATTTAGGTGACACTATAG 3´) and catalyzes the 5’ - 3’ synthesis of RNA.
The SP6 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
Assemble the following reaction at room temperature.
The volumes can be scaled according to the application.
Component | Amount |
|---|---|
Linearized DNA template | 500 ng |
Transcription buffer (10X) | 1 µl |
NTP | 1 mM each |
RNase inhibitor (20 U/µl) | 0.5 µl |
SP6 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
RNases
Activity
Proteinases
DNases
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.
Single-stranded exonucleases: 2.5 µl of the SP6 RNAP 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.
Double-stranded endonucleases: 2.5 µl of the SP6 RNAP are incubated with 0.5 µg of supercoiled plasmid DNA for 0 hours, 4 hours at 37°C and 16 hours at room temperature.
Double-stranded exonucleases: 2.5 µl of the SP6 RNAP 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.
Product Information
Storage upon receipt: Our SP6 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.
Order
Product | SKU | Pack size | Price |
|---|---|---|---|
SP6 RNA Polymerase (Glycerol-based) | Z0612 | 2000 U | Contact us |
SP6 RNA Polymerase (Glycerol-based) | Z061X | Custom | Contact us |
Do you need a quote? Contact us!