I see multiple bands after performing transcription of a large gene and letting ut run over a denaturing RNA gel. Does anyone know possible causes and how to get rid of the unwanted smaller bands?
Hi,
Is the abundance of transcript declining with increasing length? If yes, it might be a processivity issue with the particular RNA polymerase used. I briefly thought you also might have a minute contamination with an RNAse. At second thought this is less likely because you would see at least some sort of smear below the bands. Of course also the way how you prime transcription might be influencing the outcome (off-target priming) but to asses that more information would be required on the setup
Best, Ernst
Ernest is right about ghost promoters, causing secondary priming. If you are doing run off transcription then a small contaminate of uncut template can cause problems, And finally, it is also possible that the mRNA is adopting competing secondary structures. If you heat to 95 for 5 min and then cool fast on ice you might see a resolution to one band. Even 8M urea does not always melt out all RNA secondary structure.
Thank you for your answers. Here is some more info:
- The abundance of transcript is not declining with increasing length.
- I incubated 5 minutes on 65 degrees to denature RNA according to our protocol. Would increasing this to 95 remove the issue?
- repeating transcription resulted in the same bands showing up.
- The transcription is from a digested plasmid, A control digest indicates a complete digestion
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