electrophorisis of double nicked circle

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Thu Mar 23 18:17:03 EST 1995


NNTP-Posting-Host: umslts1_11.umsl.edu
Andrew Belt asks:

> I was wondering if anyone had information on the difference in mobility of
a 6000bp linear, supercoiled, or a double nicked circular DNA on a 1%
agarose gel. 

Generally ccc as it comes out of the bacteria (heavily supercoiled) will
run along side a linear a little greater than 1/2 the true linear length
of the circle.  Nicked circle runs a little slower than the linear and is
often confused with it.  EtBr slows down the circles relative 
to the linears.  The plasmid that comes out of the bacteria does not have
linear forms; the 3 bands you likely are looking at are  1x ccc, 1x nc, and
2x ccc.  There may be higher multimers.  The circular forms follow a log
linear plot higher on the gel than do the linear forms.  But during the
log-linear region, the standard curves are parallel.
I'm not sure what you mean by "double nicked" circle.  A nicked dimeric
circle runs as you would expect from the above statement.  A monomeric circle
that is nicked more than once runs just like once nicked circle.  If you
mean a linear with complementary sticky ends; it will run as a linear unless
you can keep the gel temperature below the Tm of the sticky ends; else it
will run like the nicked circle.

The exact pattern is also influenced by the temperature and salt concentration
of the gel; so you should run an uncut and a linearized control lane to clarify
your identification.  The nicked circle is the band that is very faint 
or absent immediately after CsCl banding, but which increases upon storage.
In a good cleared lysate, most of the stuff that ends up banding at the
"linear" or "chromosomal" position of the CsCl gradient is really nicked
plasmid.  Nicked circle also appears to varing degrees 
after UV treatment, or partial restriction
digestion.

Supercoiled DNA formed in vitro may vary from the heavily supercoiled position,
to the nicked circular position, and may appear as a ladder of bands.  The
exact pattern depends on the difference in conditions between the reaction 
in which the circle was closed and the gel itself. 

Hope this helps.
Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu




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