non-radioactive northern kit

Model Organism: Worms Methods & Reagents
1

I am looking for suggestions for a non-radioactive northern blot kit that has worked well. Any comments about the sensitivity of current-generation kits compared to 32P would be welcome, too. I don’t currently have a radioactivity license and want to avoid going through the hassle if I can do so without sacrificing sensitivity.

I have also heard that you can’t strip and reprobe non-radioactive northerns. Are you able to probe once and then reprobe (without stripping) with a loading control like rpa-1 that should be smaller than my band of interest?

Finally, I don’t have an X-ray film developer. I can use either our Bio-Rad ChemiDoc to scan the blot or expose it to a phosphor screen and read it with a Bio-Rad Storm imager. Anyone have experience with either of these methods?

Thanks,

Tim

2

Hi Tim,

We’ve had good results with the Roche DIG-labelling system and chemiluminescent detection. In our hands, the probes were as sensitive as 32P. The one critical parameter is to determine the amount of probe after the labeling reaction, and to use the recommended amount for the hybridization. Stripping and reprobing also works well if you use the pH-labile version.

We use X-ray film b/c that’s what we have, but it should work with other detection methods for alkaline phosphatase cleavage of CSPD.

-Harold