In article <78926422134n12 at 137.224.11.42>,
Andrea=Marconi%Algemeen%IM.WAU at Vines.WAU.NL wrote:
> >If anyone has a surefire method to isolate large plasmids from E. coli
> >I would appreciate a post to the group or an Email. I am aware of the
> >usual Kado/liu but if there is any other suggestions I would appreciate it.
> >The plasmid I am going after is about 90kb.
> >THanks in advance
>> Maybe you just tried and didn't work but I used to work with E. coli
strains transformed
> with 50-60 kb plasmids (from a gene bank) and I didn't do anything else
than the normal
> miniprep (like is described by Maniatis ) without any problem. The DNA
once extracted was
> used to transform again the same strain and was working.
>> Andrea
I find the most reliable method for detecting plasmids is still the Eckhardt
Method, which allows you to detect plasmids up to 2000 kb in size. You
will find references to various modifications of this technique in many
papers, including mine. (hynes et al. 1985, Plasmid, Hynes et al. 1989
Gene 178:111-120, Hynes
& McGregor 1990 Molec Microbiol 4:567-574). We have used it for many species
other than Rhizobium and E. coli, and find that you can isolate lots of
DNA simply ny cutting plasmid bands out of the gel. Of course you don't
get intact
DNA this way; if you want that from a large plasmid , we have found
methods such as that of Hirsch et al 1980 and Schwinghamer to be more
reproducible and less
finicky than Kado and Liu.
Michael Hynes
