Plasmid size vs supercoiling
Peter M. Muriana
muriana at aclcb.purdue.edu
Wed Jul 21 20:23:15 EST 1993
In article <44814.jdboer at sol.uvic.ca>, jdboer at SOL.UVIC.CA ("Johan de Boer") writes:
>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
>Any ideas would really be appreciated
>Johan de Boer
Think about what your saying: results from an attempted cloning experiment
are a) clones that are vector only and run ~2.5 kb (uncut), and
b) **tentative** clones that are running about ~5.0 kb (uncut), and
c) when both are cut with a Res. Enz., they are appearantly the same.
Conclusion(s): i) you've got a vector:vector ligation, linearizing with
the R.E. used for cloning gives two, same-sized products
ii) you've cloned an insert that's the same size as the
vector (less likely).
iii) you can test hypothesis (i) by seeing if cutting at
other unique vector sites keeps giving you
one (head-to-tail ligation) or two (head-to-head
ligation) digestion products.
Regards, Peter Muriana
More information about the Methods