I am trying a quick form of ribosome profiling where I digest RNA outside of harringtonine-treated samples, isolate and clean the RNA with Trizol, then use PNK modification to fix the 5'OH to 5'Phosphate for subsequent sequencing (rRNA is filtered later and the reads will be filtered for sequence size and frame). After the PNK modification (+ ATP), I have used ethanol precipitation where I add sodium acetate, ethanol, then incubate in a dry-ice ethanol bath for 25 minutes before spinning down the sample and washing the pellet with ethanol. I've noticed that after this step, I have a large, fluffy, white pellet. After resuspending this in water, the nanodrop shows a very high 260/280 of close to 7, which seems to be around where ATP would be. Can anyone recommend a way to remove this ATP, or let me know if you think this may be some other contaminant?
You can run the RNA on a denaturing PAGE gel, cut the bands with your RNA under UV light, then elute the RNA out of the bands. Another option is a spin column-based RNA cleanup kit.
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