AmpliScribe™ T7-Flash™and DuraScribe® T7In VitroTranscription Kits
- What is the longest in vitro transcript readily made with an AmpliScribe T7-Flash Transcription Kit?
While we have tested and readily produced 9-kb transcripts at Epicentre, our customers report high-quality transcripts greater than 11 kb.
- I’m using less than the recommended 1 µg of DNA template in an AmpliScribe T7-Flash reaction. How can I improve my RNA yield?
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.
- Why should I set up the AmpliScribe T7-Flash reactions at room temperature? Won’t that affect the RNA polymerase enzyme activity?
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.
- How clean does the template DNA need to be for in vitro transcription?
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.
- What is the shortest in vitro transcript readily made with an AmpliScribe T7-Flash Transcription Kit?
We have produced 26-base RNA transcripts with an AmpliScribe T7-Flash Transcription Kit.
- Can I make nonradioactive, labeled RNA using an AmpliScribe T7-Flash Kit (with labeled nucleotides or by end-labeling)?
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.
- What advantages does an AmpliScribe T7-Flash Transcription Kit have over the standard AmpliScribe High-Yield Transcription Kit?
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.
- Can I make radiolabeled probes with any of the AmpliScribe High-Yield or T7-Flash transcription kits?
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.
- I am making a template for in vitro transcription using the AmpliScribe Kit. The template structure contains a combined double-stranded T7 promoter and a single-stranded template section. Will AmpliScribe work with this template?
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.
- What is the difference between RNA made with the DuraScribe T7 Transcription Kit and RNA made with other in vitro transcription 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.
- My AmpliScribe reaction had precipitate while setting up the reaction. Is this normal?
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.
- My AmpliScribe reaction had precipitate in the reaction tube after the protocol’s recommended 30 minutes. Did my reaction fail?
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).
- What is the best procedure for cleanup/DNA template removal to recover the most RNA?
It is very simple to clean up AmpliScribe and DuraScribe reactions. We recommend three methods:
- Spin column
- Salt precipitation (using ammonium acetate with no ethanol for RNA transcripts longer than 100 bases)
- Phenol chloroform or ethanol precipitation
- What is the number of units per microliter of T7 RNA Polymerase in the AmpliScribe T7-Flash Kit?
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.
- My AmpliScribe reaction had excellent yields, but when I ran it on a native gel the RNA was much shorter than the original template. What could have gone wrong?
Single stranded in vitro transcripts are best separated using denaturing gels1. 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.
- Whenever I use my AmpliScribe Kit I get a really bad smear of RNA during gel electrophoresis instead of a band that is the size of my desired transcript. What is wrong? Did an RNase contaminant get into the reaction?
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 electrophoresis1to remove any secondary structures from the RNA and allow the RNA to migrate in a tight band rather than a smear.
1. Molecular Cloning - A Laboratory Manual, Third Edition, 2001. CSHL Press. pp 7.27 - 7.34. J. Sambrook and D. Russell.
* The use of DuraScribe™ T7 Transcription Kit to synthesise nucleic acids with non-canonical bases or for partial ribosubstitution is covered by U.S. patents 5,849,546; 6,107,037 and other patents issued or pending.
These products are accompanied by a limited non-exclusive license for the purchaser to use the purchased product(s) solely for life science research. Contact Epicentre® concerning licenses for other uses.
If you cannot find the answer to your problem below then please
contact us or telephone 01954 210 200