ZebraScribe SP6
Transcription Kit
Our ZebraScribe SP6 Transcription Kit is designed to produce high yields of ARCA-capped mRNA via in vitro transcription reaction using the SP6 RNA polymerase.
The high amount of RNA produced in each reaction (20-25 µg) is achieved by using high nucleotide concentrations with an optimized ratio of GTP to ARCA to ensure maximum yields and capping efficiency.
ZebraScribe SP6 Transcription Kit is the only mRNA kit with SP6 RNA polymerase that includes ARCA (Anti-Reverse Cap Analog) instead of a traditional cap analog (m7GpppG).
Compared to m7GpppG, which is incorporated in the reverse orientation in one third of the transcripts due to the 3′-OH of the guanosine, the ARCA includes a OCH3 modification, forcing it to be incorporated in the functional orientation. This modification ensures that 100% of the transcripts that include ARCA are properly capped and more efficiently translated.
The nucleotides and the ARCA can be provided in a convenient 5X mix, or in individual vials to allow the incorporation of modified nucleotides into the in vitro transcription reactions.
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
We recommend reading the following guide before starting:
For additional information or questions, contact us.
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
Protein expression
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 endonucleases: 2.5 µl of the SP6 RNAP are incubated with 1 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 for 0 hours, 4 hours at 37°C and 16 hours at room temperature.
Single-stranded exonucleases: 2.5 µl of the SP6 RNAP are incubated with 1 µg of ssDNA substrate (calf-thymus DNA) for 0 hours, 4 hours at 37°C and 16 hours at room temperature.
Protein expression: A linear template containing an SP6 promoter, enhanced Green Fluorescence Protein (eGFP) and poly(A) tail was transcribed and capped using SP6 RNAP and an anti-reverse cap analog (ARCA), injected into zebrafish and analyzed for eGFP expression.
Product Information
Storage upon receipt: The components of the kit stable at -20°C for up to 12 months. 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.
Order
Product | SKU | Pack size | Price |
|---|---|---|---|
ZebraScribe SP6 Transcription Kit | KT0625 | 25 reactions | |
ZebraScribe SP6 Transcription Kit | KT0650 | 50 reactions |