Screening large ccc plasmids

Paul N Hengen pnh at fcs260c2.ncifcrf.gov
Tue Jul 27 10:59:58 EST 1993


In article <1993Jul26.081619.19352 at gserv1.dl.ac.uk>
 GRGGTA at PATERSON.CHRISTIE-HOSPITAL.MANCHESTER.AC.UK writes:
 
>Does anyone have a reliable method for the isolation and electrophoretic
>separation of large (50-300 kb) ccc plasmids from bacteria?  We would like
>to screen a culture collection consisting of environmental strains of
>Vibrio and Pseudomonas spp. for large plasmids.

>Several years ago I used a method to screen large (up to 200kb I think,
>maybe more) ccc plasmids from a series of Pseudomonad plant pathogens
>which was developed for the purpose of differentiating between strains
>with some success. This involved scraping some bugs from a plate or using
>a broth culture and lysing them in situ in the wells of a vertical agarose
>gel using lysozyme and/SDS in very gentle fashion. With some practice
>reliable plasmid patterns were produced reproducably. I cannot get the 
>published references on this right now, but could get them if you are 
>interested.

Some early references are:

Eckhardt, T. 1978. A rapid method for the identification of plasmid
deoxyribonucleic acid. Plasmid 1:584-588.

Adachi, T. and V. N. Iyer. 1980. A procedure for the isolation and
purification of plasmid DNA from Rhizobium meliloti. Analytical Biochem.
101:271-274.

Kado, C. I. and S. T. Liu. 1981. Rapid procedure for detection and
isolation of large and small plasmids. J. Bacteriol. 145:1365-1373.

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA



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