Problem with precipitating RNA from ethanol solution - (Mar/30/2013 )

Hello everyone,

I'm synthesizing RNA for use as ISH probes. After the synthesis reaction, I precipitated RNA using ethanol, lithium chloride, and EDTA. I then left the samples overnight at -80, spun them for 30 minutes, removed supernatant (had a big white pellet at this stage) and washed well with 70% ethanol. After spinning down for 20 minutes and resuspending in a smaller volume, only small amounts of RNA were in the sample. When I checked the supernatant (which I kept), concentrations were high (around 200-300 ng/microliter for 100 microliter total volume). I tried spinning down again and removing the ethanol, with the same result. I'm being very careful removing the ethanol so don't think I'm removing the pellet each time. I think I may have mixed too much when I washed with ethanol.

Does anyone have any suggestions on how I could precipitate the RNA from the ethanol solution? I know glycogen can be used but am not sure how this would work with RNA in ethanol as oppsed to water. Any advice would be much appreciated.

Thank you,
Jamina

You should try increasing the concentration of ethanol. Add pure ethanol to bring the concentration up to 80% or even higher, chill, and spin down.