Plant genomic DNA digestion

James Abbott j.abbott at dundee.ac.uk
Fri Jul 10 19:04:54 EST 1998


Hi netters,

I am trying to digest genomic DNA from tobacco with BamH1 (Promega) for
Southern blot analysis, but am having trouble getting complete
digestion.  
The DNA was isolated using the Nucleon Phytopure kit, including RNAse
treatment. Uncut DNA runs as a single high molecular weight band, with
no evidence of RNA on the gel. I am using 10 ug of DNA in my reactions,
which I am trying to initially digest using 50 units of enzyme, in a 75
ul volume overnight. It is apparant that there is some cutting occuring,
with a smear being produced down to about 2 kb, however there is still a
strong band of uncut DNA, and not the normal smear you would expect to
see down to very small fragments. I've tried adding additional enzyme
and reincubating the reaction on numerous occaisons, but seem unable to
get the digestion to proceed any futher. I am aware that BamH1 is
sensitive to overlapping CpNpG methylation, but can't imagine that there
are sufficient sites of this nature in the genome to prevent apparantly
normal cutting.

Does anyone have any suggestions as to what's going on, and how to get
round this problem?

Cheers,



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