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Electrophoresis
Introduction
Electrophoresis is a separations technique that is based on the mobility of ions in an electric field. Positively charged
ions (cations) migrate towards a negative electrode (cathode) and negatively-charged ions (anions) migrate toward a
positive electrode (anode). For safety reasons one electrode is usually at ground and the other is biased positively or
negatively. Ions have different migration rates depending on their total charge, size, and shape, and can therefore be
separated.
Instrumentation
An electrophoresis apparatus consists of a high-voltage supply, electrodes, buffer, and a support for the buffer such as
filter paper, cellulose acetate strips, gel, or a capillary tube. The most popular support medium is a slab of agarose gel.
Agarose is a highly-refined form of agar, a carbohydrate obtained for sea weed. After a separation is completed the
support can be stained to visualize the separated components.
Schematic of electrophoresis apparatus
Introduction:
Agarose gel electrophoresis is an easy way to separate
DNA fragments by their sizes and visualize them. It is a
common diagnostic procedure used in molecular
biological labs.
DNA Electrophoresis:
The technique of DNA electrophoresis is based on the fact that DNA is negatively charged at neutral pH due to its
phosphate backbone. For this reason, when an electrical potential is placed on the DNA it will move toward the
positive pole:
The rate at which the DNA will move toward the positive pole is slowed by making the DNA move through an
agarose gel. This is a buffer solution (which maintains the proper pH and salt concentration) with 0.75% to 2.0%
agarose added. The agarose forms a porous lattice in the
buffer solution and the DNA must slip through the holes
in the lattice in order to move toward the positive pole.
This slows the molecule down. Larger molecules will be
slowed down more than smaller molecules, since the
smaller molecules can fit through the holes easier. As a
result, a mixture of large and small fragments of DNA
that has been run through an agarose gel will be separated
by size. This is a graphic representation of an agarose gel
made by "running" DNA molecular weight markers, an
isolated plasmid, and the same plasmid after linearization
with a restriction enzyme:
http://faculty.plattsburgh.edu/donald.slish/Gelmov.html
These gels are visualized on a U.V. trans-illuminator by
staining the DNA with a fluorescent dye (ethidium
bromide). The DNA molecular weight marker is a set of
DNA fragments of known molecular sizes that are used as
a standard to determine the sizes of your unknown
fragments.
If you click on the link you will see a short video that simulates the movement of the DNA bands through the gel.
When looking at the video, note that bands of a low molecular weight move very quickly through the gel while high
molecular weight bands move very slowly.
Interpretation:
Much information can be derived from this gel. As you read the text below, refer back to the figure.
By looking at the migration of the DNA molecular weight standards, you can tell that the migration of DNA through
an agarose gel is not linear with respect to size.
Graphing:
Measure the distance in mm from the bottom of the origin (wells) dashed line in the gel diagram above to the bottom
of each DNA marker fragment in the gel, and record it below. Then, plot the relationship on the semi-log graph
paper provided. What is the relationship between the distance migrated and the molecular weight of each band?
Mobility of DNA marker fragments in agarose gel electrophoresis:
Marker size (kilobases)
23.0
9.4
6.6
4.5
2.3
2.0
0.56
Distance migrated (cm)