electrophorisis of double nicked circle

David Gonda david_gonda at qm.yale.edu
Thu Mar 23 19:19:11 EST 1995


In article <s911870-2303951451410001 at umslts1_11.umsl.edu>,
s911870 at umslvma.umsl.edu (Andrew Belt) wrote:

> 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.  Any information would be beneficial

I have much experience with this from my previous life working with recA
protein and the defined DNA substrates used for recA-mediate pairing
reactions.

The mobilities of supercoiled ("RFI"), nicked circular ("RFII") and linear
("RFIII") can be very persnickity. *In general*, in a 1.8% gel, if you run
it quickly (that is, at 150V for an approx 16 cm long gel in TAE), then
RFI will migrate the fastest, followed by RFIII, with RFII having the
lowest mobility. However, about one time out of every 10, the mobilities
would all switch so that, for example, RFIII would run the fastest, and
supercoiled the slowest, for absolutely no good reason that anyone could
figure out (my best guess was that it had something to do with the depth
of buffer over the gel, but I never did anything systematic to sort it
out). You do not get good separation in a 1.8% gel if you run it slowly
(say, 20V overnight for same size gel). Conversely, a 1.0-1.2% gel will
*usually* give you very good separation of RFI, II, and III if you run it
slowly (say 20V overnight); run it quickly and everything tended to mush
together. However, again, every now and then mobilities would change,
though the slow low percentage gels seemed to be much more consistent than
the fast high percentage gels. The take home lesson we all came away with
was that if we *really* cared which band was RFI vs. RFII vs. RFIII, and
we *really* cared that we could separate them (an usually we did *really*
care), we ran the appropriate standards on the gel to make sure we could
interpret it correctly (say, a fresh supercoiled DNA prep for RFI, an
older supercoiled prep that had sat out a bit for RFI + RFII, and DNA cut
at a unique restriction site for RFIII).

By the way, we worked with DNA from filamentous phages, which were around
the size you're concerned about (e.g. M13 = 6407 bp), so your mileage
shouldn't vary *too* much from the above. Hope this helps...

-- 
David Gonda 
Yale University 
Dept of Molecular Biophysics and Biochemistry
david_gonda at qm.yale.edu
[the opinions above, which are mine, are only mine, and belong to me]



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