rcaspi at sdcc3.ucsd.edu (Ron Caspi) wrote:
>Hi, is there a TOL plasmid expert out there?
>>I am working with a strain of P. putida, and would like to check for
>the presence of such a plasmid. Does anybody have a protocol that will
>let me visualize such a plasmid on a gel? (unfortunately, pulse field
>is not available).
>>Many thanks in advance
I did my Masters thesis on TOL plasmid detection at SDSU (yes, right
accross town). My thesis should be available at the SDSU library. Since
TOL is a low copy number plasmid, the Eckert (sp?) method of in-well
lysis is grossly inadequate, since it doesn't give a strong enough signal
to really make working with the plasmid possible. If you want a TOL
plasmid prep protocol use a modified Anderson and McKay (1983) Appl & Env
Microbiol. 46:549-552. The prep was originally designed for isolating
large plasmids out of lactic Streptococci, but it does wonders in
isolating TOL plasmids. If you want TOL plasmid DNA sufficiently purified
(no RNA) you will need to follow-up the plasmid prep with CsCl density
centrifugation (CsCl centrifugation strait from a culture lysate will
give you a pathetically low plasmid yield). If you want the
modifications, let me know [give me a fax #]. Its important to grow up
the P.putida w/ TOL in a modified media to enrich for TOL plasmids as
well (containing m-toluate and 4-methyl benzyl alcohol). TOL plasmid
containg bacteria have a nasty habit of losing their plasmids (or having
them transpose to the genome) if they are not being used.
Good luck. TOL plasmids are easily one of the toughest plasmids to work
with in Molecular Biology. If you want PCR primers for the plasmid, I
have some sequences that seem to work well [amplifying a region of the
xylB upper pathway gene] that I never got around to publishing. I am
doing work TOTALLY unrelated to TOL plasmids since graduate school, so
you are welcome to the (old) information.
Talk to Barbara Hemmingsen at SDSU. She was my thesis advisor. I'm not
sure whether she has had students continue my work or not.
Neenah, WI 54956
E-mail: kkorth at kcc.com