In article <3v2mq7$lot at mserv1.dl.ac.uk>, Karine dronen
<Karine.Dronen at im.uib.no> wrote:
> I'am gone use Pseudomonas putida in exogenous plasmid isolation. I want
to use the Eckhardt
> procedure fore screening, hoping to catch the big plasmids.
Unfortunately this Pseudomonas strain
> seems very hard to get lysed with lysozyme, RNase and protease
treatment. So if there is anyone
> out there who had tried Pseudomonas strains with the Eckhardt technique,
please shear your
> experience with me. Also: If there is another successful method for
screening Pseudomonas in big
> scale, I would appreciate hearing from you.
>> Thank you,
> Karine Dronen
>> e-mail address: Karine.Dronen at im.uib.no
I have in fact used Eckhardt gels a lot. Mostly with Rhizobium (various
species)
Agrobacterium, and E.coli. My students have done Bacilli and Butyrivibrio.
I have tried Pseudomonas (now Burkholderia) solanacearum succesfully, and
been able to see small plasmids in P. aeruginosa. The key seems to be to
use a very
small number of cells. Grow them in media which they do not grow particularly
well in (nutrient poor). try playing with the SDS concentration as well.
For your info we use horizontal gels, containing 1% SDS (final conc.) this
precludes the necessity of using double well systems etc.
For details see Hynes et al. Plasmid (1985) and more recently Hynes and
McGregor (1990) Molec. Microbiol. 4: 567-574.
Good luck
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