poor random priming for Northern Blot probes - (Aug/17/2006 )
I am a newbie when it comes to Northern Blots and had a problem with making my radio-labeled probe. I used the Random Primers kit from Invitrogen to P32 label a 1.3kb DNA fragment that had been gel isolated and purified. I followed the directions word-for-word (which may have been my first problem ). My problem is that I got half of the cpms that the kits says you are supposed to get (10^4 compared to 10^8). I'm wondering if I shouldn't have put the probe through the spin column (from Ambion) to clean it up. Any ideas?
Also, what is the usual way to strip a blot? My membrane is postively-charged nylon.
Thanks for the help!!
i elute the purified probe by 200µl twiceand that increases the recovered probe without effetc on hybridization (as400µl against 10ml are very few...)
for stripping : i put boiling 1%SDS on the membrane and let cool to RT.
Rinse with prehybridization buffer and that's ok.