I eluted my RNA after the RPE wash during the RNeasy kit and realized that my sample has carried over a lot of ethanol. How do I remove it? Should I re-precipitate or just let the ethanol evaporate? How do I re-precipitate to get rid of the ethanol in my final sample without losing too much yield.
Hi Aditi
Some amount of ethanol in the sample can be easily removed by evaporation....but if it is goo amount you can easliy reprecipitate it.....To avoid loss of RNA in reprecipotation you can also use a carrier molecule such as 'yeast tRNA' which is commercially available from standard companies supplying molecular biology grade chemicals
bye
Aditi,
+1 Precipitate your RNA (add to your sample 0.1 vol of 3M sodium acetate and 2.5 vol EtOH, mix well, keep overnight at -20C, centrifuge at 4C). After you have washed your pellet with 75% EtOH, you might want to wash your sample once more with 95% EtOH to facilitate drying of the sample. After you have aspirated 95% EtOH, centrifuge again briefly, and aspirate the remaining ethanol. Then leave the open tubes at room temperature for a short while (5-10 min) to evaporate the residual ethanol (but do not overdry!!!), and then dissolve your RNA in TE or RNAse-free H2O. Usually, we do not loose much yield.
In addition...you can add to the dr. Mashanov recipe 3 µl of LPA (Linear Polyacrylamide) as carrier to precipitate small RNAs and in order to avoid RNA loss...
hope it helps...
In RNAeasy kit, before eluting your RNA at the last step, just keep your column in new tube and dry your coulum for 5 min at full speed in centrifuge. It removes the ethanol retained in your column. If it is still present there, just call to customer care of the kit manufacturer. usually it should not be like that, otherwise no one will use these commercial kits.
Centrifuge the spin column at ≥12,000 x g for 2 minutes in the emptied Collection Tube to ensure complete removal of the wash buffer.
You can rapidly evaporate off small amounts of ethanol from an RNA sample by heating at 65 degrees for 5 minutes, in a heating block with the lid open
RPE buffer, I assume you are using a Qiagen RNeasy kit? There is a step before elution to centrifuge the spin column at ≥12,000 x g (by itself with no buffers) for 2 minutes in the emptied Collection Tube to ensure complete removal of the wash buffer. All the liquid that comes through the flow through are residuals of EtOH. Carefully handle that spin column and transfer the filter to a new collection tube without touching the flow through liquid and elute.
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