Can 0.22 µm filters get rid of RNases? - (Oct/06/2005 )
I am having a heck of a time with RNA electrophoresis on 1% TBE gels, and I am suspecting RNases are at fault. I am using DEPC water and RNaseZap, but was wondering if anyone knew if filtering my buffers through a .22ul filter (usually for sterilizing cell culture medium) would help? Thanks :
A 22 micron filter will not get rid of RNases. If you're samples are treated with an anti-RNase and you're using DEPC water, then perhaps your buffer is contaminated?
I'm assuming that you're having problems with smearing, right? Are you circulating your running buffer during the run?
i don't think that even if the buffer was contaminated, RNases would not have time to efficiently degrade RNA. Moreover the lodaing buffer is often with formamide, which do not allow a good rnase activity.
Regarding my work, the more quick is the extraction the better the quality is. and always work on ice for that...
But 0.22µm will not help. Use fresh solutions.
Ya, my ladder is one big smudge. I am not circulating the running buffer, would I need a pump for that? Thanks for your help, no filtering for me today. But I am making all new buffers. How do you decontaminate your electrophoresis aparatus? I have found a lot of different ideas.. would love to know about one that really works. Thanks!
you can find some tips about washing your gel box with a 0.1N NaOH, then with DEPC H2O
I do a couple brief washes with 70% EtOH (easier and less caustic than lye), let evaporate, rinse a couple times with clean water. This works for me. I suppose it depends on how paranoid you feel you need to be...I also have a dedicated gel box that only gets RNA stuff and dedicated pipettors and all that stuff...if you use the same gel box for all sorts of other protocols you will have to work harder to decon before running RNA, right?
BTW, I have not found "RNAse away" to work any better than wiping your countertops with 5% bleach, then a quick wipedown with 70% EtOH before starting work. This is about the cheapest way to keep everything all nuclease-free and stuff
as far RNA electrophoresis is concern it is usually recommended in MOPS -DENATURATING GEL (formaldhyde). Prepare 20x MOPS in DEPC treated water and then autoclave it. You also need to properly treat your electrophoresis tank. First wash your electrophoresis tank with detergent. then rinse with ethanol and then fill it with 3% Hyperogen peroxide for half an hour. then rinse with DEPC Water.
Hope it will work for you.
Regarding circulating your buffer... you can use a pump or a pipettor to circulate the buffer a bit every half an hour or so. I definitely notice a difference when I do or do not do this, but I suppose it depends on which buffer you're using.
Also, I'd DEPC treat and bake any spatulas or mortar/pestle's you're using to extract your RNA.
are you sure that you ladder is OK ? Is anyone else in your lab using tyhe same ladder and not getting a smear ?
You could have two problems - one being sample preparation - perhaps the hardest bit - the other being that your tube of ladder is already degraded
and therefore that your problem isn't related to actually running the gel (cleaning the gel tank etc)
hope you get it sorted
thanks so much to everyone for their helpful replies. With a combination of all your hints, I was fortunate enough to run two beautiful gels on Monday with lovely clear ladders. Many many thanks, I really appreciate your help.