Agarose Gel Electrophoresis

Agarose gel electrophoresis is a physical method to determine the size of
DNA. In this method, DNA is forced to migrate through a highly cross-linked
agarose matrix as a result of an electric current. In solution, the phosphates on
the DNA have negative charges, therefore the molecules migrate to the
positive (red) pole. The three factors which affect migration rate through
a gel are size of the DNA, conformation of the DNA, and ionic strength
of the running buffer. Another crucial material is the TBE which is normally
used as a running buffer and have a constant ionic strength constant during
electrophoresis. Electrophoresis is considered to be as a sieving process. The
larger the fragment of DNA, the more easily will it become entangled in
the matrix and, therefore, the more slowly will it migrate. Small fragments,
run more quickly than large fragments at a rate which is proportional to their
size. The relationship of size to migration rate is linear throughout most of
the gel, except for the largest fragments since they take longer time to
penetrate the gel, they do not have a linear relationship rate to the size.. The
matrix can be adjusted, though, by increasing the concentration of agarose
(tighter matrix) or by decreasing it (looser matrix). A standard 1% agarose
gel can resolve DNA from 0.2 - 30 kb in length. Most of the DNA
’
s that we will be examining are plasmids .Plasmid DNA can exist in three
conformations: supercoiled, open-circular, and linear.
In vivo 
, plasmid DNA is tightly supercoiled circle to enable it to fit inside the cell. ,
most of the DNA remain supercoiled, but a certain amount can sustain single-
strand nicks. Given the presence of a break in only one of the strands, the
DNA will remain circular, but the break will permit rotation around the
phosphodiester backbone and the supercoils will be released. A
small, compact supercoiled knot of DNA sustains less friction against the
agarose matrix than does a large, floppy open circle. Therefore, for the
same DNA. .