While we have tested and readily produced 9-kb transcripts at Epicentre, our customers report high-quality transcripts greater than 11 kb.
With lower concentrations of DNA template, you can increase RNA yields by increasing the reaction time from the recommended 30 minutes up to 2-4 hours, depending on the amount of template used. Increasing the reaction temperature from 37°C to 42°C also improves the yield.
The components of the reagents in a standard 20-µl reaction are very close to their solubility limits. If you set up the reaction on ice, some reaction components (such as the buffer and the nucleotides) will precipitate. After components precipitate, warming the reaction tube only partially resolubulizes the reagents. The AmpliScribe T7-Flash Enzyme Solution is added last, so it may be kept on ice until needed.
Very clean template DNA ensures the best performance of an in vitro transcription reaction. If using a PCR product for the template, purify the desired product from the reaction to remove any remaining primers, primer-dimers, and residual dNTPs. If you are using a plasmid for the template, in order to transcribe the desired RNA product, completely linearize the plasmid, leaving a blunt or 5´-overhanging end. Uncut plasmid serves as excellent template, but the RNA polymerase will transcribe past the desired transcription stop point and can continue around the plasmid several times before the reaction finally stops, creating an RNA that is far longer than desired and includes undesirable vector sequences.
We have produced 26-base RNA transcripts with an AmpliScribe T7-Flash Transcription Kit.
Yes, you can directly incorporate derivatized nucleotides (with moieties like Cy5, biotin, or digoxygenin) into the transcripts or you can perform post-transcriptional labeling of purified RNA transcripts at the 5´ or 3´ ends. Please contact us for specific protocols.
The two main advantages of the AmpliScribe T7-Flash™ Kit are: a) improved RNA yields, even better than the excellent results obtained with the AmpliScribe High-Yield Transcription Kits; and b) a fast, 30-minute procedure.
Yes, but not recommended. While rarely done any more, radioactive probes may be made using post-transcriptional labeling. Generating radioactive RNA during the in vitro transcription reaction requires a lot of radioactive nucleotide due to the high concentrations of radioactive NTPs required to prepare probes with high specific activity. This is extremely expensive and potentially dangerous. You can prepare radioactive RNA probes by using alkaline phosphatase to generate a 5´-hydroxyl end, followed by radioactive tagging using γ-32P-ATP and T4 Polynucleotide Kinase, or by using α-32P-(5´,3´)-bisphosphate NDPs and ligating to the 3´ end of the RNA using T4 RNA Ligase.
While reports of successful hybrid templates for in vitro transcription have been reported, you should use fully-double stranded templates for the best results when transcribing RNA using AmpliScribe and DuraScribe kits.
The DuraScribe T7 Transcription Kit produces RNA that contains nucleotides with a 2´ ribose fluorine and that is resistant to degradation by A-type RNases (like the RNase found on human skin), while the AmpliScribe T7-Flash and other transcription kits make standard RNA. DuraScript™ RNA can be reverse-transcribed, like regular RNA, and can be digested by RNase III. However, DuraScript RNA cannot be used as a template to produce proteins by in vitro translation.
AmpliScribe reaction set-up is temperature-sensitive and loss of yield may occur if you use cold reagents when assembling the reaction. All components, including enzymes, should be brought to room temperature to minimize precipitate formation. If precipitates form while setting up your reaction, re-solubilizing may be incomplete. This may impact downstream RNA yield. We suggest running an in vitro transcription reaction using the control template to confirm yields are within kit specification.
Successful AmpliScribe reactions may result in formation of a white precipitate. This precipitate is the synthesized RNA from the reaction – the yields can be so high that the RNA precipitates out of solution. Don't worry – simply dilute the reaction using RNase-free water and purify using any of the recommended methods (ammonium acetate precipitation, spin column, or ethanol precipitation).
It is very simple to clean up AmpliScribe and DuraScribe reactions. We recommend three methods:
Unfortunately, this is considered proprietary. AmpliScribe T7 Polymerase solution contains additives that enhance transcription, so the number of units per AmpliScribe reaction is not comparable to the number of units used per reaction with pure T7 RNA Polymerase.
Single stranded in vitro transcripts are best separated using denaturing gels. Denaturing gels allow in vitro transcripts to separate on the basis of their length rather than based on their length + secondary structure. When using native gels, sample migration may be altered by secondary structures in the transcripts.
No. It is likely that your electrophoresis was performed on a native gel, which does not remove any secondary structures from the RNA. Use denaturing conditions for electrophoresis to remove any secondary structures from the RNA and allow the RNA to migrate in a tight band rather than a smear.