DNA ELECTROPHORESIS
Gel electrophoresis is a method that separates macromolecules-either nucleic acids
or proteins-on the basis of size, electric charge, and other physical properties. Gel
electrophoresis is an important molecular biology tool. DNA sequencing, fingerprinting,
and genetic engineering are based upon it. Gel electrophoresis separates DNA fragments
by their size or molecular weight. The agarose gel acts like a sieve, separating different
sized fragments while the electric current provides the driving force. DNA, a negatively
charge molecule, is attracted to the positive electrode; the voltage determines how fast the
DNA will travel through the gel. Larger molecules or DNA fragments become entangled
in the gel and travel more slowly, while smaller ones pass through more easily and travel
farther down the gel.
DNA gels are made of agarose, a highly purified agar, which is heated and
dissolved in a buffer solution. The agarose molecules form a matrix with pores between
them. The more concentrated the agarose, the smaller the pores. We will be using a 0.8%
agarose gel because we are looking at large DNA fragments thousands of base pairs in
length. A 2% agarose gel separates DNA fragments, which differ in length by as few as
50-100 base pairs. Fragments of linear DNA migrate through agarose gels with a mobility
that is inversely proportional to the log10 of their molecular weight. In other words, if
you plot the distance from the well that DNA fragments have migrated against the log10
of either their molecular weights or number of base pairs, a roughly straight line will
appear. Circular forms of DNA migrate in agarose distinctly differently from linear
DNAs of the same mass. Typically uncut plasmids will appear to migrate more rapidly
than the same plasmid when linearized. Additionally, most preparations of uncut plasmid
contain at least two topologically-different forms of DNA, corresponding to supercoiled
forms and nicked circles.
Several factors have important effects on the mobility of DNA fragments in agarose gels,
and can be used to advantage in optimizing separation of DNA fragments. Chief among
these factors are:
Agarose Concentration: By using gels with different concentrations of agarose, one can
resolve different sizes of DNA fragments. Higher concentrations of agarose facilite
separation of small DNAs, while low agarose concentrations allow resolution of larger
DNAs.
Voltage: As the voltage applied to a gel is increased, larger fragments migrate
proportionally faster than small fragments. For that reason, the best resolution of
fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to
the gel (the cm value is the distance between the two electrodes, not the length of the
gel).
Electrophoresis Buffer: Several different buffers have been recommended for
electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Trisacetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat
different rates in these two buffers due to differences in ionic strength. Buffers not only
establish a pH, but provide ions to support conductivity. If you mistakenly use water
instead of buffer, there will be essentially no migration of DNA in the gel. Similarly, if
you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in
the gel to melt it.
Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates
between bases of nucleic acids and allows very convenient detection of DNA fragments
in gels. As described above, it can be incorporated into agarose gels, or added to samples
of DNA before loading to enable visualization of the fragments within the gel. As might
be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and
therefore its mobility.
Preparing an Agarose Gel
Buffer solution: 1X TBE buffer: (89 mM Tris-base, 2 mM EDTA, and 89 mM Boric
Acid).
Agarose gel: Weigh 0.8 grams of agarose powder and place it in a 125 or 250 ml flask.
Add 100 ml of 1X TBE buffer to the flask. Swirl to mix the solution.
Boil in microwave oven until the solution is completely clear and no small floating
particles are visible. Swirl the flask frequently to mix the solution and prevent the agarose
from burning. Cool the solution to 55C before pouring the gel into the plastic casting
tray. Higher temperatures will melt the plastic tray. Place the plastic comb in the slots on
the side of the gel tray. The comb teeth should not touch the bottom of the tray. Pour the
agarose mixture into the gel tray until the comb teeth are immersed about 6 mm or 1/4"
into the agarose. Push any bubbles to the side farthest from the wells.
Allow the agarose gel to cool until solidified. The gel will appear a cloudy white color
and will feel cool to the touch (about 20 minutes). Gels can be stored, wrapped in plastic
wrap, in the refrigerator for a few days.
Loading and Running an Agarose Gel
1. Remove the comb from the wells by pulling straight up the comb. Be careful not to
tear the wells as you remove the comb. Place the gel tray in the gel box with the wells
closest to the negative (black) electrode.
2. Add enough 1X TBE buffer to fill the buffer tank and submerge the gel about 1/4
inch.
3. On a piece of wax paper, mix 5 µl of gel loading dye with 10 µl of your DNA sample.
Mix the solution by pipetting the dye up and down into the 10 µl DNA sample. The
gel loading dye contains glycerol which will make your DNA more dense so that it
will sink into the wells. It also contains dye molecules which are smaller and travel
faster through the gel than the DNA molecules. The dye molecules provide a visual
tracking method so you know how far the DNA has traveled through the gel.
4. Make a sketch of the gel; include all of the wells and the positive (red) and negative
(black) electrodes. Indicate under each well the name of the sample to be loaded.
5. Pipette 10 µl of the first sample, usually a reference sample such as a 1 kb ladder, into
the first well. Keep the tip of the pipettor ABOVE the well. The DNA will sink into
the well because it has been mixed with loading dye. Load the next sample into the
next well. continue until all of the samples and controls have been loaded into the gel.
Remember to record on your sketch the order the samples were loaded (including
standards). Close the top of the box. Plug the leads into the gel box.
6. Plug the other end of the leads into the power source and turn it on. Run the gel at
between 80-120 volts until the loading dye has traveled 1/2 - 3/4 of the way down the
gel (about 30-45 minutes).
7. Turn off the power supply before opening the gel box.
Staining the DNA
The DNA must be stained in order to be seen. DNA can be stained with fluorescent or
chemical dyes. Research laboratories use ethidium bromide, an ultraviolet (UV)
fluorescent stain, because it shows very small amounts of DNA and is faster to use.
Ethidium bromide, however, can cause cancer and mutate DNA. DNA can be stained
with methylene blue, a chemical dye, which binds to DNA.