Agarose Gel Electrophoresis

A method of gel electrophoresis used in biochemistry and

molecular biology to separate DNA or RNA molecules by size.
This is achieved by applying an electric field to move the
negative charged nucleic acid molecules through
an agarose matrix. The separated DNA may be viewed with
stain, most commonly under UV light and the DNA fragments
can be extracted from the gel with relative ease.

Factors affecting Migration

A number of factors can affect the migration of nucleic acids.

These include

1. Size of DNA or RNA

Smaller molecules travel faster than larger molecules in gel.

2. Voltage

The higher the voltage, the faster the DNA moves. But voltage
is limited by the fact that it heats and ultimately causes the gel
to melt. High voltages also decrease the resolution (above
about 5 to 8 V/cm)

3. Gel Concentration

Agarose gel has greater range of separation and is therefore

used for DNA fragments of usually 50-20,000 bp in size. The
concentration of gel affects the resolution of DNA
separation.Larger molecules are resolved better using a low
concentration gel while smaller molecules separate better at
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high concentration gel. High concentrations gel however
requires longer run times.

 1% gel is common for many applications.

 0.7% gives good separation or resolution of large 5–10kb
DNA fragments
 2% gives good resolution for small 0.2–1kb fragments.

The concentration is measured in weight of agarose over

volume of buffer used (g/ml).

4. Buffer

In general, the ideal buffer should have good conductivity,

produce less heat and have a long life. The most common
buffers used for agarose gel include

 TAE: Tris/Acetate/EDTAand
 TBE: Tris/Borate/EDTA.

TAE has the lowest buffering capacity but provides the best
resolution for larger DNA. This means a lower voltage and more
time, but a better product.

General procedure

Casting of gel

The gel is prepared by dissolving the agarose powder in an

appropriate buffer, such as TAE or TBE, to be used in
electrophoresis. The agarose is dispersed in the buffer by
heating. The melted agarose is allowed to cool sufficiently
before pouring the solution into a cast as the cast may warp or
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crack if the agarose solution is too hot. A comb is placed in the
cast to create wells for loading sample and the gel should be
completely set before use.

Loading of samples

Once the gel has set, the comb is removed, leaving wells where
DNA samples can be loaded. Loading buffer is mixed with the
DNA sample before the mixture is loaded into the wells. The
loading buffer contains a dense compound, which may be
glycerol or sucrose that raises the density of the sample so that
the DNA sample may sink to the bottom of the well. The
loading buffer also includes colored dyes such as bromophenol
blue used to monitor the progress of the electrophoresis.

Electrophoresis

Agarose gel electrophoresis is most commonly done

horizontally whereby the gel is completely submerged in buffer
during electrophoresis. The buffer used in the gel is the same
as the running buffer in the electrophoresis tank.

Staining and Visualization

The most common dye used to make DNA or RNA bands visible
for agarose gel electrophoresis is ethidium bromide (EtBr)
which intercalates into DNA and fluoresces under UV light. By
running DNA through an EtBr-treated gel and visualizing it with
UV light, any band containing more than ~20 ng DNA becomes
distinctly visible. EtBr is a known carcinogen, however and
safer alternatives are available e.g. SYBR Green, GelRed etc

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Applications

 Estimation of the size of DNA moleculesfollowing

restriction enzyme digestion, e.g. in restriction mapping of
DNA.
 Analysis of PCR products, e.g. in molecular genetic
diagnosis or genetic fingerprinting
 Separation of restricted genomic DNA prior to Southern
analysis or of RNA prior to Northern analysis.

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