Enzyme or probe - who's the trouble maker in my southerns? - (Sep/08/2006 )
I'm struggling a bit with the establishment of DIG southern blotting - at this point, I get a smear with numerous light bands rather than one strong band.
I have already shortened the probe to 300 nt, and when I blast it, there is no significant homology anywhere else in the mouse genome. Therefore, I think it might be the restriction enzyme which I am using: Spe l. Does anybody have any negative or positive experience with this enzyme in Southern digests?
My DNA seems to be ok, as I got the probe for 5' end screening to work (using EcoRl digest).
Any comment welcome - thanks a lot for your help!!!
I presume you've tried to alter the hybridization temperature and the wash stringency. Your probe could be very high GC. These sometimes give rather unselective binding.
SpeI is a pretty forgiving enzyme.