Genomic Digest Difficulties

IAN WATSON iwatson at metz.une.edu.au
Mon Jul 19 04:53:53 EST 1993


I'd better start right at the begining:

I am an honours student studying an anerobic bacteria which causes footrot in
sheep known as Dichelobacter nodosus (Formerly Bacteroides nodosus). I am
specifically studing two strains from the H serotype, one virulent and the other
benign. I have a heaps of trouble growing these H strains as a broth culture,
so, I grew them on TAS plates and suspended the cells of the plate and carried
out a genomic DNA prep. I acheived a good yield, however, whenever I try to 
digest them (with EcoRI and HindIII) They do not digest at all. Initially I
thought that there may be proteins present in the prep or perhaps phenol which
was interfering with the digest. So I carried out a phenol extraction again
and ethanol precipitated them again. After this the digest failed again. So I
thought that there was an outside chance that the TE buffer that the genomic DNA
was dissolved in had too high a concentration of EDTA causing the nucleases to
fail so I ethanol precipitated them again and then dissolved in new TE. However,
This also failed. It has been suggested to me that I geneclean the preps and
then digest them but I feel that genecleaning such large fragments might cause
damage to the genomic DNA. I've tried to think of everything but nothing seems
to work. I have considered carrying this procedure out again but I dont see the
point in spending time on a time consuming procedure such as this just to end up
getting the same results again. 

I have also considered that there may be methylation, however, this is unlikely
due to other strains I've worked on (which I've been able to grow in a broth and
have had no trouble with) have not shown any sign of resisting being digested.

I really need some suggestions on this one because I've run out of ideas and
I'm also running out of time.

Any suggestions would be extremely appreciated.

			Ian Watson

			iwatson at metz.une.edu.au  

			Dept of Biochemistry, Microbiology and Nutrition
			The University of New England
			Armidale 2351



More information about the Methods mailing list