I made a T7 transcription in the presence of 5-Bromouredine (50% 5-BrU and 50% of uridine). How to determine which part of synthesised RNA has bromouridine and how to calculate how many BrU are included per one RNA molecule?
T7 transcription yeld was lower than usially (without BrU) around 10 times.
Hey,
I don't think there is a way you can determine that, because it's a statistically process so it's different for every single molecule. Chances are that you don't have a 50/50 ratio either because of different incorporation rates for Uridine and 5-BrU, but I don't know that from the top of my head.
You could try the T7 variant Y639F for better yield. The Mutation is usually used to incorporate 2'-modified pyrimidines, but as far as I know it also helps with some other modified nucleic acids.
Cheers,
Florian
Florian, thank you. I am thinking about measurment of the UV absorption spectra. I guess, BrU has absorbance different to uridine.
can you provide details of your experiment? is it an in vitro synthesis? if so, are you adding nucelotides to the reaction? 5-BrU only or mixed with uridine?
JP
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