Southern Blot: which restriction enzyme? - (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!!!
Are you sure that the enzyme does not cut somewhere in the sequence that you are probing for (maybe star activity)? That is the only reason that I can see that should give you multiple bands, 300nt should be pretty specific