RNAse protection assay - No bands (Feb/21/2005 )
I'm doing a RNAse protection assay but I never see protected bands on the gel only background, even in the negative control i still have background. The sequence was sequenced twice, and the are antisense to the probe I want to see, the Tm of the shortest protected band was calculated (74°C) and I incubated the hybridisation mixture o.n. @ 65°C. And the probes are not degraded.
Did anyone have the same problems and could solve them finally?
Is your probe good? Are you running just the probe in a gel lane? If the probe is cruddy sometimes all ya get are shmeers..... I am an expert in bad probe makin'!!
PS We hybridize at 56c overnight.....but then again we use a purchased DNA template set
Oh....yeah...I just notice that you said that your probes are not degraded...sorry .....in that case.....I don't know why .......all I can say is change out your reagents......Some of them can get kinda picky for RPA's....