genomic DNA digest - (Aug/20/2004 )
I am trying to digest mouse genomic DNA for a southern blot. When digested with either HIndIII or NdeI, I get a nice smear as expected. But when I digest with either speI or BglII (neither one of which is methylation sensitive) I get a thick band at the top of the gel, and hardly any smear at all. I have tried both NEB and Promega's enzymes, both new, with 500ng of DNA with 20 to 25 Units of enzyme for 3Hours+ or overnight. Does anyone know any issues related to those enzymes? or why one digest would work and not another????
It's just mouse gemone has fewer cutting sites of some enzymes. I've tried EcoRI, BamHI, HindIII and PstI. The latter (HindIII and PstI) are much more efficient than the former.
BTW, you only use 500ng DNA for the Southern? What kind of labelling method do you use? I had trouble while using DIG for mouse genome Sothern. I usually use more than 20ug DNA. I am wondering whether I used too much DNA.
I use 20ug for a southern, but I was just testing the different enzymes on 500ng DNA first.
Most protocols I've seen use at least that much and sometimes more.
I've been told to make sure the probe works well first with standard P32 protocol. Then try DIG labelling.
I am digesting 10ug genomic DNA for southern blot using Sca I . However since I am using an external probe for hybridisation I am not getting my band which is approx 10 kb lenght .
if any one knows about this would you please shed some light .