T7
RNA Synthesis Kit
Our T7 RNA Synthesis Kit is designed to provide flexible, high yield synthesis of RNA using in vitro transcription with T7 RNA polymerase. The synthesized RNA can be used for downstream applications such as RNA structure and function studies, microinjection, transfection, in situ hybridization and in vitro translation.
The kits are available in 25, 50 or 200 reactions, with each reaction yielding around 100-120 µg of RNA after a 2-hour incubation.
Components included in the kit are: a T7 RNA polymerase enzyme mix, individual NTP solutions, reaction buffer and control DNA template. DNase I and EDTA are included for removal of the DNA template after the reaction.
The enzyme mix includes our ArtPure™ T7 RNA polymerase, 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 | Recommended amount or volume |
|---|---|
Linearized DNA template | 0.1-1 µg |
10X reaction buffer | 2 µl |
ATP | 1.5 µl |
CTP | 1.5 µl |
GTP | 1.5 µl |
UTP | 1.5 µl |
Enzyme mix | 2.5 µl |
Nuclease-free water | To 20 µl |
*0.1 µg of PCR product or 1 µg of restriction digest template.
Incubate the reaction 2 hours at 37°C.
To remove the DNA template, add 1 µl of DNase I and incubate at 37°C for 15 minutes.
Inactivate the DNase I by adding 2 µl EDTA (50 mM), 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.
Full-length transcripts (%): Amount of full-length transcripts produced relative to amount of total RNA, calculated using the area under the curve (AUC) from electropherogram obtained with a 2100 Bioanalyzer RNA 6000 Nano kit.
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 T7 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 T7 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 T7 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.
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 |
|---|---|---|---|
T7 RNA Synthesis Kit | KS07025 | 25 reactions | |
T7 RNA Synthesis Kit | KS07050 | 50 reactions | |
T7 RNA Synthesis Kit | KS070200 | 200 reactions |