In article <3jjh78$gc at styx.uwa.edu.au>, Kirsty Townsend wrote:
> Dear Fellow Micro-Netters,
>> I'm hoping someone out there can help me, because I'm having huge problems
> with restriction digests of particular strains of Pasteurella multocida.
> They are known to have a high LPS content in the cell wall, but I thought
> I was allowing for that during the million ether extractions. But the DNA
> I get at the end of it all, will cut with Eco RI but not with Pst I. I
> have tried using increased enzyme concentrations, different buffer etc
> Thanks in advance,
>> Kirsty Townsend
We had a similar situation with one of our strains of Rhizobium leguminosarum
where we we couldn't cut the DNA with Pst. It turned out that that strain
(and that strain alone) carried a plasmid which had on it a gene coding
for a methylase that protected PstI sites from digestion. We have cloned
this
enzyme and the associated restriction enzyme by a methylase protection
procedure. If your DNA cuts with EcoRI and a large number of other enzymes
but not with Pst, I would suggest that you probably also have a methylase.
The best solution is probably just to use other enzymes in the meantime.
Michael Hynes
