RNA contamination w/ ethanol - (Jan/13/2006 )
Hmm...I might be having this problem. Does clean-up with a Qiagen column help get rid of ethanol contamination?
I tend to run a little on the paranoid side about my RNA samples...after decanting, I leave the tubes inverted on a test-tube rack and I put them in the TC hood, with the blower on but no light (and a note asking others not to turn on any UV )
I find that it takes around two hours or so for them to dry completely...I got in a hurry once and resuspended them a bit early, got a bunch of rotten real-time data (the spec readings weren't great but I went ahead anyways because I really needed the data to tie up a paper) after all the work! so yeah, John is totally right, makes a big difference downstream
To answer my own question - the column must get rid of ethanol, right?! Large amounts of ethanol are used during Qiagen purification process, so it can't be something that sticks to the column before elution..
With a column it is very common to get ethanol carryover in your samples
As a precuation you should always do an extra 2 minutes spin step between the second RPE wash and the elution step. This is usually enough to remove most of the residual ethanol, however I very often still see reduced 260/230 ratios using this method compared to ChargeSwitch which is completely aqueous
Great, thanks for the tip. I'm having trouble with low PCR efficiencies, and I didn't realize this could be a contributor. I was taught in grad school to only let my RNA dry for about 10 minutes before resuspending - I didn't realize you can let it dry for 2 hours! Is it difficult to resuspend if you wait that long? In addition, I have been using the Qiagen column for all my samples as well. So.. this could potentially be a big problem for me. I will also look into using ChargeSwitch.
Thanks again, guys!
Ok I am checking out ChargeSwitch - do you use it to clean RNA or cDNA? Or both?
Soluene - if you are using chargeswitch this may not apply; I do not know anything about that product?
however, I allow my RNA pellets to dry until they are, um, dry. I don't worry about how long it takes, I leave them be until I can't see any fluid upon close inspection, and then I wait another 10 or 15 minutes to be sure. I do it in the TC hood because I figure it's the cleanest place in our lab and there's good airflow; it's not precisely nuclease-free but I think they're less likely to pick up nucleases there than anywhere else in the lab and I have not had a big degradation problem
when I go to resuspend, I just put in a little TE, vortex briefly. then, I either let them sit at RT about 5 or 10 min (or on ice up to an hour, depends on my schedule) then I give them a quick spin and go to use them. this has always been sufficient for dissolution
Great thanks for the tip. I was taught a while back that RNA is very difficult to dissolve if you let the pellet dry completely. Maybe that is only in DEPC water? I dissolve mine in TE buffer as well, though. I'll have to give this more thought because I don't think I could claim a hood for a 2-hour time block (we have about 50 lab people here!) without someone going in it with lots-o-nucleases.
oright guys, updates on my re-precipitation:
it seems like it was an ethanol contamination because after I finished, I let it dry for over an hour, although I was advised not to in fear of crystalization and inability to resuspend RNA....and I don't know if this helped or done the opposite, but I actually after an hour still could see some droplets so I put the tube under microscope (light on o fcourse) and I saw the ethanol droplets rising slowly along the wall of tube... kinda cool
so maybe heat helped it... Do you think I should do this all the time from now on? but I might have denatured some of my RNA because I had a small yield: 1.05 micro g (A 260 = 0.042 and A 280= .02) so maybe not a good idea, or I had little RNA to start with anyway (I used most of my sample testing)..... but does my sample seem very pure? or are my #s too small to rely on?
You've all been helpful, thanks again!