plasmid size v supercoiling

harvey at afrc.ac.uk harvey at afrc.ac.uk
Thu Jul 22 03:43:00 EST 1993


> Hi,
> 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?
>
> Any ideas would really be appreciated


You have generated a head-to-tail dimer, this is not uncommon, trimers are
rarer. They persist because your bugs must be Rec-. Alternatively, your insert
is same size as vector.




Regards

Harvey
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HARVEY at UK.AC.AFRC.JII
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