RNA:sharp bends and a smear - (May/10/2010 )
I`m trying to isolate RNA from mouse liver tissue using TRI Reagent. Everything looks fine (OD 260/280 as well as OD 260/230 are quite good), until electrophoresis. I manage to get two sharp bends of 28S and 18S rRNA, in a right proportion, but between these two bands there is some kind of a smear.
Also, I have intense background signal, and sometimes it appears that my whole gel is glowing. I`m using EtBr in a loading buffer (50% glycerol, 1mM EDTA, 0.4% bromphenol blue, 1mg/mL EtBr) and I don`t destain my gels.
What am I doing wrong?
There are a bunch of different size RNAs (think about all the different proteins encoded and their range of sizes), if you extract enough you will be able to see it on a gel. 1 mg/ml is very high for EtBr, try 0.5-1 ug/ml in the gel (or post stain at similar concentrations).