Hello everyone,
I have an odd problem. I generated different templates for in vitro transcription using T7 RNA polymerase. While I had nice results for 2 of 4 templates last year using ambion MegaShort Skript Kit, now I cannot seem to repeat these results. Due too higher yield and the easier handling, I switched from linearized plasmids to PCR products as templates. The templates look fine in gel analysis after PCR and have a length between 150 and 250 bp.
I tried transcription using different polymerases from different companies in their according buffer and using the according protocol. I am downscaling to 10 / 20 µL, which I did before as well.
Interestingly, the resulting templates show very weak bands of correct size and strong bands at 100 nt less than expected in denaturing Urea PAGE for all 4 templates.
Nucleotide concentration is checked and high enough. Lower transcription temperatures (down to 20 °C) did not show any different results.
Any advice is highly appreciated!
Thank you!
Hi Janina, to my experience, RNA Polymerase sometime falls off while generating small sized RNA transcripts rather than the full length. To my suggestion, linearized plasmids after phenol-chloroform precipitation is far better than using PCR product as template. How long are you incubating? I have read that overnight incubation at 37 Degree for smaller size transcript is highly recommended. You may try so. Best wishes :)
Dear Sharmin,
Thank you for your answer. I have tried as well 3h, 4h and over night - no difference. I read about falling off as well, but then I would expect a smear rather than clear and strong bands of consequently 100 nt too short. Also I read falling off happens for long transcripts. I don't think that's the cause
Hi Janina Luders,
I am facing similar issue with the in vitro transcription. What I get is a smear from PCR amplified template. Using linearised plasmid as a template, I could see two bands on Urea PAGE (one of the expected size and the other one was slow migrating probably due to secondary structure less likely though).
Any suggestions, please share.
Thank You very much in advance!
Dear Janina,
Did you find a solution for this issue?
I am experiencing very similar problems, tried two different T7 enzymes (Roche and Ambion) without success.
I am using high quality PCR products as templates (400-500 bp in size, full T7 promoters on both sides to generate dsRNA), after transcription there are very small amounts of smaller size products and nothing at all at the right sizes. Would appreciate if you could share your outcome from this troubleshooting.
Thank you,
Rodrigo
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