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Problem with precipitating RNA from ethanol solution


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#1 genotype

genotype

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Posted 30 March 2013 - 05:21 PM

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

#2 phage434

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Posted 30 March 2013 - 05:36 PM

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.




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