2 replies to this topic

[marc_U7snurp]

Hi everyone,

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?

Thanks  

[labprincess]

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'!!  

Labprincess


PS We hybridize at 56c overnight.....but then again we use a purchased DNA template set

[labprincess]

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....



Labprincess