RNA isolation: ethonal wash step - Do you resuspend the RNA pellet by pipetting? (Jan/20/2006 )

Hello,

during RNA isolation after the precipitation with isopropanol, you remove the supernatant and add 1 ml 75% ethanol to the tube for the wash step.

How far do you go in resuspending the RNA pellet?

Do you just add ethanol and proceed to centrifugation?

Or do you add ethanol and vortex (in my case, the pellet detaches from the tube wall, but remains solid and will not dissolve by any means)?

Or do you add ethanol and really resuspend the pellet by pipetting till it is unvisible?

So, what do you think is the best way to get pure RNA?

Thanks for suggestions, Tobias

I did never try if it is possible at all to "solve" the pellet in 75% ethanol, I would fear it just gets unnecessary fragmentation. So what I do is most often to just add the ethanol and repeat the washing and in cases it is really important to have little salt (electroporation) to pipette carefully a bit so the pellet detachs from the wall but stays intact.

I asked the same question in another thread, and this is the advice I got.. (posts are at the end of the thread)..

http://www.protocol-online.org/forums/inde...showtopic=12473

I just do a brief vortex to sorta break up the pellet and let it swirl around in the ethanol and then spin.

No matter how hard you try, you're unlikely to get your RNA resuspending fully in 75% EtOH. You'll just end up creating a colloid by breaking the pellet into thousands of pieces to small to see. Whilst this isn't a major problem it does mean you have to follow it with a fairly lengthy centrifugation step (say 30 mins at 14000 rpm) in order to get a pellet again.

My suggestion would be to vigourously vortex for about 10 seconds (as most people suggest) but do this up to 4 or 5 times. Each time the impurities in the pellet will leach out. This also means your pellet will stay intact - hopefully - so you only need to do a 2 min centrifugation step between each wash.

You might also try to cool your EtOH in a -20 deg C freezer. You can then vortex for longer.