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Gel Electrophoresis

-method which separates molecules according to their weight and structure using
an electric field applied to a gel matrix

Principle:

DNA molecule fragments are introduced into a gel matrix, and an electric
field is applied across the gel. Because DNA molecules are negatively charged, they
will move through the gel toward the positive pole of the electric field. As different-
sized DNA fragments move through pores in the gel matrix, longer DNA fragments
encounter more resistance and move more slowly than shorter fragments. The
bands farthest from the wells contain the shortest DNA fragments, and the bands
closest to the wells contain the longest fragments.

• Agarose gel- a highly purified agar, heated and dissolved in a buffer solution.
The agarose molecules, when cooled, form a matrix with pores between
them. The more concentrated the agarose solution, the tighter the matrix
and the smaller its pores.
• Glycerol- make the sample dense
• Tracking dye: Bromphenol blue
• Running Buffer: TAE-establish a pH and provide ions to support conductivity.
• Ethidium Bromide- a fluorescent dye that intercalates between bases of
nucleic acids and allows detection of DNA fragments in gels under ultraviolet
light.

Factors affecting migration:


1. DNA conformation (circular or linear) - circular plasmids will appear to
migrate more rapidly than the sample plasmid when linearized.
2. Agarose concentration - higher concentrations of agarose facilitate separation
of smaller DNAs, while low agarose concentrations allow resolution of smaller
DNAs.
3. Voltage- as the voltage applied to a gel is increased, the larger fragments
migrate proportionally faster than small fragment. The best resolution of
fragments larger than about 2kb is attained by applying no more than 5 volts
per cm to the gel.
4. Electrophoresis buffer- DNA fragments will migrate at somewhat different
rates on two buffers with different ionic strength.
Fig.8.1 DNA bands illuminating under UV light stained with ethidium bromide after gel
electrophoresis