How to remove traces of ethanol after RNA wash - (Jul/21/2006 )
I've got a dilemma: The trade-off between over drying the RNA pellet by sucking off completely the 70% ethanol. (thereby disturbing the fragile RNA pellet)
Leaving a little 70% ethanol behind(sacrificing purity and not disturbing the RNA pellet). I did the latter. After taking the (abs) reading and running an 2% agarose gel, the concentration of the RNA does not tally with the brightness of the 18s and 28s bands.
Question is how to remove the traces of ethanol in the RNA solution? I read that ethanol will inhibit reverse transcription and give spurious results.
after decanting the spnt, I invert my open tubes in a very clean spot with little lab-air-flow (our little tissue-culture hood, or in the 37C incubator, which is disinfected) and leave them for about 30min-1 hour (if it's 37C) or about 1.5-2 hours (if it's room temp)
this allows evaporation and I generally get pretty solid ratios and clean bands. I check the tubes every 15 min or so and resuspend the pellets in tris as soon as they are completely dry (to the naked eye).
does this help? I'm sure others have other methods?
i rinse my RNA sample with 80% Ethanol (made by DEPC treated water) and then invert my open tubes in a clean spot (on kimwipe tissue paper) and i left it in the RNA prep room for 5-10 mins.
Try precipitating the RNA solution again with 0.25M - 0.3M (final conc.) Potassium acetate and 2 vol etOH (or a vol isopropanol). -20C for not less than 1 hr. Precipitation steps and washing with etOH. As for drying, you could try the advice given above.
Oh ya, make sure the ratio of Ab260/280 and 260/230 are acceptable. I don't think EtOH contam will affect the RNA spectro reading. Note if dilution factor was taken into consideration.
I use a glass pipette pulled into a fine capillary and suck off most of the remaining EtOH, then aior dry for about 5-10 minutes at room temp.
after spin i pipett the etoh, briefly spin again in order to decant etoh, pipett it and allow 37° for 104 to remove residual ethanol.