I am a chem grad student. Now I am doing RNA stuff. My question is "how do you check the RNA purity after transcription".
We transcripted RNA from ds-DNA which has T7 promoter, and digested excessive DNA with DNase I (RNAse free). After phenol extraction, cutting 8% denaturing PAGE gel to separate target RNA. And then continued with elution, ethanol precipitation, dissolved in DEPC treated water.
When checked the purity of final product, we got problem: In denaturing PAGE gel, there was alway significant band tail in the lower mobility direction, but the control sample of pure RNA and its DNA template only gave a sharp, clean band. (see attachment).
I am pretty sure that I cut the gel right for the purification of RNA. So where did the tail come from? The protein should be removed by phenol extraction, right? And the templated DNA also should be digested by DNase I (37oC, 30 min).
I know that some people using agarose gel for purification, but the resolution of PAGE gel should higher than agarose (synergel).
Could you please give me some suggestions about how to get rid of this RNA band tail?