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Practical No 14 : Agarose Gel Electrophoresis

Objectives : To understand the principles and to, become knowledgeable of the practical
aspects of Agarose Gel Electrophoresis

Introduction:

Unlike protein which is analyzed by polyacrylamide gel electrophoresis, nucleic acids are analyzed by
agarose Gel Electrophoresis. The nucleic acids are larger molecules, and therefore, require a medium
with bigger pores for their separation. The pores formed in acrylamide gels are too small for the
separation of large nucleic acid molecules. However, polyacrylamide gels are used in certain procedure
such as DNA sequencing. The agarose is polymer of carbohydrate purified from certain species of
seaweeds. The solid agarose is solubilized in hot electrophoresis buffer and poured into a gel casting
chamber. Upon cooling the solution from a solid matrix with randomly oriented polysaccharide
polymers, with randomly oriented polysaccharide polymers, which create pores for migration of nucleic
acid molecules during electrophoresis.

Materials
An electrophoresis chamber and power supply
Gel casting tray
Well forming combs
Tris-acetate-EDTA (TEA) electrophoresis buffer (40 mM Tris-Acetate. 1mM EDTA, pH 8.0)
6X gel loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol in water),
ethidium bromide (10mg/ml in water)
UV trans-illuminator (an ultraviolet light-box)

Procedure
Preparation of Agarose Gel
First 30 m1 of a 1% (v/w) solution of agarose was made in TAE buffer in a conical flask and the flask
was weighed. Then the mixture was heated to boiling in the microwave oven. The flask was examined
and continued heating till the agarose was fully dissolved. The container was weighed and distilled
water was added to compensate for the loss off weight after boiling. The gel was allowed to cool for few
minutes at room temperature before pouring the gel. Then 2µl of ethidium bromide solution was added
to the agarose solution. Then the gel casting chamber was prepared, making sure that the edges are in
place firmly against the ends of the casting tray. The agarose solution was poured into the casting tray,
being careful not to overflow the tray. Finally the comb was placed and the gel was left to cool and
solidify.

Preparation of the samples


An appropriate volume of DNA sample was mixed with 1/5 volume of loading buffer.

Loading and running the gel


The wedges were removed from the casting tray and the buffer reservoir was filled with TAE buffer
until the buffer was 1-2 mm deep over the gel. The comb was carefully removed by lifting it straight out
of the gel slowly. Each sample was carefully pipetted into a well in the gel. After the all the samples
have been loaded; the leads were connected from the power supply to the gel box. Making sure the
electrodes were oriented correctly (wells at negative end; DNA will run to the positive end). The output
level was set to 80 volts and the power turned on. The gel was run until the tracking dye xylene cyanol
reaches the edge of the gel. The gel was placed on the trans-illuminator and the clear plastic shield was
placed over the trans-illuminator window before turning on the UV light. Finally the trans-illuminator
was turned on and the gel was observed. Confirm the presence of DNA (orange bands) and turn off the
trans-illuminator.
Observations

Clear DNA bands were observed in the gel after the electrophoresis once exposed to UV light, as shown
in the below image.
Discussion

The separation of DNA molecules by agarose gel electrophoresis is similar to the separation of proteins
by SDS-PAGE electrophoresis. In both cases, the molecules to be separated are loaded onto a gel and
then subjected to an electrical field. The cathode (negative electrode) attracts positively charged
molecules, while the anode (positive electrode) attracts negatively charged molecules. This attraction
causes the molecules in the mixture to migrate through the gel at different rates depending on their size,
shape, chemical composition, and electrical charge. As the molecules migrate at different rates, they
gradually separate from each other.
The main difference between protein electrophoresis and DNA electrophoresis lies in the support
medium. While polyacrylamide gels are generally used for protein separations, agarose gels are
commonly used for DNA separations. Agarose is a polysaccharide that is extracted from seaweed.
Agarose gels are cast by dissolving agarose in a Tris-buffered solvent at a high temperature, and then
pouring the solution into a horizontal tray and allowing it to cool. A comb is inserted into the cooling gel
so that wells are formed as the gel solidifies. The cooled gel is then transferred into a gel box where it is
submerged in a Tris-buffered electrode solution before loading DNA samples into the wells. Since these
gels are run horizontally and beneath the electrode buffer, they are sometimes referred to as “horizontal
gels” or “submarine gels”.

DNA molecules will migrate towards the anode during electrophoresis because they have a high
concentration of negatively-charged phosphate groups in the sugar-phosphate backbone of the double
helix. These negative charges provide a fairly uniform charge-to-mass ratio among all DNA molecules.
Because of this uniform ratio, the migration rate of DNA molecules during agarose gel electrophoresis is
almost entirely a function of size. As with protein electrophoresis, smaller molecules move through the
gel more rapidly than larger molecules. Scientists generally measure the size of DNA molecules in terms
of number of base pairs (bp) rather than molecular weight (MW).

DNA, like most proteins, is colorless. Therefore, before DNA samples are loaded onto an agarose gel,
they are mixed with a sample buffer that includes a blue tracking dye. During electrophoresis, the
tracking dye migrates very rapidly through the gel, along with the smallest of DNA fragments. When the
dye approaches the end of the gel, you know it is time to stop the electrophoresis. Glycerol is also
included in the sample buffer in order to make the sample denser so that it settles into the well as it is
loaded.
After electrophoresis is complete, the gel is stained so that the DNA bands can be seen. The most
commonly used dye for staining DNA is ethidium bromide. DNA bound to ethidium bromide fluoresces
strongly under ultraviolet light, so the gels are viewed and photographed using ultraviolet light.
However, because both ethidium bromide and ultraviolet light are mutagens, you will be using a less
sensitive but safer stain for your DNA gels: methylene blue. Methylene blue is a blue-colored stain that
binds to DNA in the same way that Coomassie Blue binds to proteins. The blue color is easily seen
using normal visible light.

Figure 01: Methylene blue stained DNA gel patterns

During SDS-PAGE electrophoresis there is a linear relationship between the migration distance of the
proteins and log of MW. Similarly, during agarose gel electrophoresis there is a linear relationship
between the migration distance of the DNA molecules and log of bp (number of base pairs.) Because of
this, agarose gel electrophoresis can be used to estimate the size (number of base pairs) of DNA
molecules. In order to do this, a standard curve that shows the relationship between migration distance
through the gel and log of bp must be generated. The standard curve is generated by measuring the
migration distance of several DNA molecules of known size (number of base pairs), plotting a scatter
diagram of migration distance vs. log of bp, and then using linear regression to determine the equation
of the “best fit” straight line for the data points. Once this is done, you can substitute the migration
distance of any DNA and can find its size and finally the number of base pairs.
Electrophoresis: a non-selective approach is defined as a molecule with a net charge will move in an
electric field. For example, any charge molecule subject to an electric field will move.

Electrophoresis depends on: Net charge of protein, applied electric field and Friction

To conduct electrophoresis we use gels. There are many differences between these two gels:
Agarose

Linear polymer composed of alternating isomers of the sugar galactose (D- and L-galactose). polymers
aggregate to form supercoiled structures of a radius of 20-30 nm and variable length (around 800
galactose residues).standard agaroses melt at ~ 90 ºC and gel at ~ 40 ºC; gelation results in mesh of
channels with diameters from 50- >200 nm (see figure) Agarose gel is used for larger molecules but It
does not identify small differences between two molecules bands. It contains large pores.

Figure 02: Agarose gel structure

Agent responsible for cross linkage

Methylene bis acrylamide is an agent that is used to produce cross linkages. Acrylamide polymerizes the
gel. Benefit of cross linkage is to pass large molecules easily from the pores. DNA is negatively charged
and therefore migrates to the anode (positively charged electrode), if a voltage is applied.

Buffer composition

the salt content (ionic strength) of the electrophoresis buffer will influence migration; without salt the
DNA will barely move; in the presence of two much salt, the conductivity will be very high and the
produced heat will impair separation; we will use a Tris-borate buffer (pH 8.0) that contains EDTA
(TBE); EDTA is a chelating agent that binds divalent cations such as Mg+2 that many nucleases require
for their activity; EDTA thus protects the DNA from enzymatic degradation

Gel loading buffer, added to DNA sample

 contains glycerol or sucrose to increase density (otherwise, sample would dissolve in running
buffer and not sink into the gel pocket)
 contains dyes that facilitate observation of the sample during gel loading and

Electrophoresis
 bromophenol blue: runs about as fast as 300-bp DNA (linear!); separation is usually stopped
shortly before this dye reaches the end of the gel (depending on the size of the fragments that
need to be recovered)
 xylene cyanol (optional): runs about as fast as 4-kb DNA

Ethidium bromide staining of DNA

 Ethidium bromide is a fluorescent dye that binds to DNA (intercalates between the stacked
bases); binding increases fluorescence about 10-fold
 when irradiated with UV light of a wavelength of 302 nm, ethidium bromide will emit
fluorescence light of a wavelength of 590 nm (orange)
 the dye can be included in both the running buffer and the gel, the gel alone, or the gel can be
stained after DNA separation (which comes in handy if you forgot to add the dye before running
the gel); to minimize ethidium bromide-containing waste, we will include the dye only in the gel
at a concentration of 0.5 mg/ml (added to the buffer used to dissolve the agarose

Disadvantage: ethidium bromide is positively charged and will migrate in the opposite direction as the
DNA, which leads to an uneven background staining) Ethidium bromide is a strong mutagen and should
be handled with care. Ethidium bromide-containing gels will be treated as hazardous waste.

Figure 03: Ethidium bromide chemical structure

References

1. Dawson, C.R. and Magee, R.J. (1995): Plant tyrosinase (polyphenol oxidase). In: Colowick S
Kaplan NO (Eds) Methods in Enzymology. Academic Press New York 2 : 817 - 827
2. Doyle, J. J. and Doyle, J. L. (1990): Isolation of plant DNA from fresh tissue. Focus. 12:13-15.
3. Brody, J.R., Kern, S.E. (2004): History and principles of conductive media for standard DNA
electrophoresis. Anal Biochem. 333(1):1-13. PMID 15351274
4. Primrose, S. et.al. Principles of Gene Manipulation. Oxford: Blackwell Science, 2001.
5. Milan Bier (ed.) (1959). Electrophoresis. Theory, Methods and Applications (3rd printing ed.).
Academic Press. pp. 225. LCC 59-7676. OCLC 1175404.
6. Robyt, John F.; White, Bernard J. (1990). Biochemical Techniques Theory and Practice. Illinois:
Waveland Press. ISBN 0-88133-556-8.
7. HHMI Undergraduate Research Studio - Freshmen Biology Section, Fall 2007