On Friday, November 8, 2013 11:59:39 PM UTC-5, DK wrote: > This might be quite a silly question but just in case: > > > > I will be making tRNA in vitro using T7RNAP. The in vitro reaction > > contains crazy amounts of nucleotides (10 mM each NTP). What > > is the best way of cleaning up such reactions to remove salts and > > nucleotides? Without losing the precious RNA, of course. > > > > Had it being DNA > 200 bp, I'd know what to do - either columns > > or EtOH precipitation would do fine. I am unsure however what I > > can expect from 85 bp tRNA. > > > > For EtOH pption: How long to keep on ice? (Is low temp even > > needed; for DNA I find that it is not beneficial at all). What cation > > works best for small RNA? Also, some protocols suggest wash > > with 95% EtOH to prevent loss. Good or not? The concentration is > > expected to be high, > 0.1 mg/ml although the volume for now > > is small, only 20 ul. Any benefit from adding linear polyacrylamide > > under such conditions? > > > > How do standard silica columns work for small RNAs? Anything needs > > to be changed for binding? How does the capacity compare to > > plasmid DNA? > > > > Any other relevant advice is greatly appreciated. Thanks! > >
Most silica-based binding columns do not perform well on small RNAs such as tRNA, at least not without modification. Several vendors sell columns for micro-RNA purification, which should work, although it might be overkill, expense-wise, since those kits will also include reagents you won't need for a simple clean up. Note that the trick to getting small RNAs to bind is a (most likely proprietary) additive added to the binding buffer, not the columns itself. As indicated, ordinary sodium acetate/ethanol works just fine to precipitate tRNA; in fact, in some procedures, tRNA is often used as a carrier for precipitation. Nick _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
