Plasmid size vs supercoiling
mkennedy at chmeds.ac.nz
Thu Jul 22 17:26:04 EST 1993
In article <44814.jdboer at sol.uvic.ca>, jdboer at SOL.UVIC.CA ("Johan de Boer") writes:
> We are trying to isolate a recombinant molecule that consists of about 3.5
> kb vector and 2.5 kb insert. When we run the (uncut) plasmid DNA from
> minipreps on an ethidiumbromide/agarose gel the major band runs at a
> certain position, but one clone runs much slower (at approximately 2.5
> and 5 kb linear markers respectively). The faster ones turn out to be
> just the vector, when we linearize the DNA with an enzyme that cuts
> one of the cloning junctions. The slow running DNA would look promising,
> however it looks exactly the same as the others after linearizing it.
> Is it possible for a plasmid from two colonies to have such different
> supercoiling patterns?
> Has anyone seen this before?
Plasmids often form dimers, which can't be resolved unless a specific
site-specific recombination mechanism exists to resolve the dimer. So yes, you
can get dimeric and trimeric forms of plasmids (some of these account for the
extra bands you see in some uncut gel resolved plasmids, in addition to linear
and open circ forms). In fact, if you cut these out of a gel and transform
them back into a bug, they should continue to propagate as dimers. If I
locate my PhD thesis, and dust the cobwebs off it, I'll provide some refs
about this phenomenon; it is well documented in the literature on plasmid
replication and partition.
NNNN NN Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz) ZZZZZZZ
NN NN NN Cytogenetic and Molecular Oncology Unit ZZZ
NN NN NN Christchurch School of Medicine ZZZ
NN NNNN Christchurch, New Zealand ZZZZZZZ
Phone (64-3)364-0880 Fax (64-3)364-750
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