GEL ELECTROPHORESIS

A. Theory
Since the introduction of gel electrophoresis as a means of studying nucleic acids some 10
years ago, this method has become a power and versatile tool in the investigation and
characterization of DNA molecules. It is rapid, precise, inexpensive and requires only small
amounts of DNA. Gel electrophoresis allows the rapid separation and high resolution of DNA
species on the basis of molecular size and conformation. It is therefore an extremely useful
method for the estimation of molecular weights of both intact plasmid molecular and linear
DNA fragments produced by restriction endonucleolytic cleavage, provided one has standards
of known length.
With respect to the sizes of the DNA molecules to be separated, either polyacrylamide
or agarose is used as the gel material. Only agarose gels will be discussed here since the
resolution capacity of this method is sufficient in most cases.
Agarose is a polysaccharide extracted from various red algae. When DNA is subjected
to agarose gel electrophoresis, it is forced to migrate through the interstices of this network
toward the anode (due to its negatively charged phosphate residues) with a migration velocity
depending on the followings
• Molecular weight of DNA
• Conformation of DNA (CCC, OC, Linear)
• Pore sizes of the gel (determined by the agarose concentration)
• Voltage gradient employed to the gel
• Electrophoretic buffer
All these parameters interfere with another, but there are some general guidelines, like for
example;
1. High voltage gradients are employed for separating small DNA fragments. Better
resolution of high molecular weight DNA is archived at low voltage gradient
2. Small pore size (between 0,7 and 1 % agarose) are used for small DNA fragments (up to
107) Large DNA molecules (15-40 x 106) are usually submitted to electrophoresis in low
concentration gels (0,3-0,5 % agarose)
Agarose gels can be prepared at concentrations ranging from 0,3-2 % (w/v). Either
vertical or horizontal slab gels can be used. Horizontal gels are more convenient to handle and
more stable, especially at agarose concentration lower then 0,8 %, although it has been
reported that vertical gels give sharper bands. Gels are prepared by suspending and boiling the
calculated amount of agarose powder in an electrophoretic buffer until the solutions is
completely homogenous and clear. The molten agarose is cooled to 50-60° C before pouring
into the tray. After the gel is completely set, samples are loaded after mixing with loading
buffer. To increase the density, loading buffer contains glycerol or sucrose (to a final
concentration of 5-10 %). Addition of ficoll (1-2 %) avoids the formation of U-shaped bands.
A tracking dye may be added as a visible marker; usually bromophenol blue is used at a final
concentration of 0,025 %.
Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested form
them for use). Because they cut within the molecule, they are often called restriction
endonucleases. A restriction enzyme recognizes and cuts DNA only at a particular sequence
of nucleotides. For example, the bacterium Hemophilus aegypticus produces an enzyme
named HaeIII that cuts DNA wherever it encounters the sequence
5′GGCC3′
3′CCGG5′
The cut is made between the adjacent G and C. This particular sequence occurs at 11 places in
the circular DNA molecule of the virus phiX174. Thus treatment of this DNA with the
enzyme produce 11 fragments, each with a precise length and nucleotide sequence. These
fragments can be separated from one another and the sequence of each determined.
HaeIII and AluI cut straight across the double helix producing “blunt” ends. However,
many restriction enzymes cut in an offset fashion. The ends of the cut have an overhanging
piece of single stranded DNA. These are called “sticky ends” because they are able to form
base pairs with any DNA molecule that contains the complementary sticky end. Any other
source of DNA treated with the same enzyme will produce such molecules. Mixed together,
these molecules can join with each other by the base pairing between their sticky ends. The
union can be made permanent by another enzyme, DNA ligase that forms covalent bonds
along the backbone of each strand. The result is a molecule of recombinant DNA (rDNA).
Restriction enzyme digested DNA is usually run in Tris-acetate or Tris-borate buffer can
be used (largely dependent on plasmid size). Nevertheless, it is necessary to adapt
electrophoretic conditions to the specific circumstances of the particular experiment.
DNA bands can be visualized by standing with ethidium bromide (EtBr). The sensitivity
of this staining technique depends on the concentration of the DNA applied to the gel. The
less the amount of DNA, the weaker the fluorescence. Gels can be stained either by including
EtBr in the gel (Note: EtBr influences the mobility of DNA to small degree) or after the run
by soaking it in EtBr solution (0.5 μl / ml) for 30-60 min. To diminish the background of
fluoresce, it is advisable to destain the gel with redistilled water, if the second way is
preferred. DNA bands are than detected by irradiation with UV at 254 nm on a
transilluminator. (Note: Irradiation with sort wavelength UV causes damage to the DNA and
destroys its function). The illuminated gel is photographed, usually with a Polaroid camera,
equipped with a red filter to exclude UV light. DNA fragments may have the same
electrophoretic mobility as the marker dye (depending on agarose concentration and voltage
gradient). Thus, these DNA bands may be missed since bromophenol blue absorbs
fluorescence released from DNA-bound EtBr and obscures the DNA.
As mentioned above the mobility of DNA in gels is a function of its molecular weight
and within a limited range inversely proportional to its size; the lager size, the slower the rate
of migration. Thus agarose gel electrophoresis allows the determination of relative and
approximate molecular weight on the basis of electrophoretic mobility in relation to the
mobility of DNA standards of known sizes. Mobilities are usually calculated from
photograph. Accuracy of the measurement can be improved by photographic enlargement.
The distance from the sample to the top of each band is measured (Migration Distance) and
the relative mobility is calculated (Migration Distance divided by the length of the gel). A
standard curve is constructed by plotting the log of molecular weight of standard against their
relative mobilities and drawing a best fit line through these points. In the small size range,
linear relationship is obtained. However, migration of very large DNA molecules become
more or less independent of size, so that the relationship is no longer linear and a curve is
observed at the top of the scale. Molecular weights of the samples are determined by the
placing the measured mobilities on the standard curve and interpolating the size.

B. Experimental
1. Materials
TE (Tris-EDTA) Buffer: 10 mM Trizma Base
1 mM EDTA (pH: 8.0)
Tris-Acetate Buffer: 40 mM Trizma Base
20 mM Acetic Acid
2 mM disodium EDTA (pH: 8.0)
EtBr Solution: 10 mg/ mL
Loading Buffer: 0.25 % Bromophenol blue
0.25 % Xylene cyanol
15 % ficoll
 40 % (w/v) sucrose

WARNING: Ethidium Bromide is a powerful mutagen. Use gloves when working with it.

2. Procedure
1. Dissolve 0.4 g of agarose powder in 50 mL 1X Tris-Acetate buffer (0.8 % w/v; gel size ~
8 x 10 cm)
2. Cool the solution down to 50-60° C and add 4 μL 10 mg/ml EtBr solution
3. Insert the gel into the apparatus and fill the apparatus with 1X Tris-Acetate buffer.
4. Run the gel at 90 V until the tracking dye reaches to the end of the gel (for about 1 hour)
5. After electrophoresis, gel stain the gel is visualized on UV transilluminator (if EtBr is not
included in the preparation of the gel, stain the gel with 0.5 mg/mL EtBr solution for 15 min
and than destain for 10 min. with deionized water prior to visualization under UV radiation).
1. Draw the bands seen in the gel on the UV transilluminator.

WARNING: The transilluminator emits short wave UV light which will damage skin and
eyes, during prolonged exposure and be sure that proper shielding is in place before turning
on the transilluminator.

7. Draw the calibration curve and calculate the molecular weight of the unknown fragment.