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Agarose Gel Electrophoresis
Description
An electrophoresis technique that is used to separate DNA fragments by size.
Negatively charged DNA fragments are separated in an agarose gel bed by
subjecting them to an electric field. The gel is stained so that the DNA bands can
be visualized. The DNA fragment sizes are determined by comparison to a set of
size standards.
Uses:
Quantitative and Qualitative Analysis, Characterization, Purification
• Determination of sizes of DNA fragments when compared to standards
• Evaluation of purity of DNA after DNA isolation (DNA and RNA contaminants
can be observed).
• Determination of DNA size following restriction enzyme digestion.
• Purification of DNA fragments after separation by recovery from the gel.
• Separation of DNA fragments prior to further characterization and analysis
(for example Southern Blotting).
Detailed Description:
Agarose gel electrophoresis is a wonderful tool, and the workhorse of the
Biotechnology lab! The theory is simple. DNA molecules are long polymers, and
the size of the strand is proportional to its negative charges because of the
phosphate backbone. The longer the DNA fragment, the greater its charge. Thus,
when placed in a semi-permeable buffered media, DNA will migrate by size, in a
rate roughly proportional its charge to mass ratio, toward the positive electrode.
Fragments will separate during the course of the gel run, with larger fragments
migrating more slowly. Following the separation, which is usually monitored by a
tracking dye such as bromophenol blue, the gel is stained, the bands visualized,
and a photo or digital record of the gel made. Analysis of a gel will always elicit a
cry or excitement, or a sigh of disappointment. Gels are easy to run, but it is also
easy to make mistakes. Many scientists have pieced together broken gels after
they dropped the slippery gel on the floor.
The sizes of the unknown DNA bands are determined by comparing them to a
set of DNA markers, frequently referred to as “ladders” because of the rung-like
appearance in the gel lane. A number of markers, in various sizes, are available
commercially.
The separation is dependent on the type of agarose gel, the concentration of
agarose used to make the gel, the buffers, amount of voltage used, temperature
and the size of the DNA. A typical gel would be 1% agarose in a buffer such as
Tris-Acetate/EDTA, run at 80 volts for 60 minutes. Higher percentage gels are
used to separate smaller fragments. Specialty gels are used to separate very
small DNA pieces, or for other applications, such as using low-melt gels for
recovery of DNA. Lower voltages, coupled with longer running times, provide
optimum resolution, such as that required for Southern Blots or forensic
applications. Pulsed-field electrophoresis can be used to separate very large
DNA fragments.
The most common stain is ethidium bromide, which intercalates into the doublestranded DNA (and some RNA) strands. With ultraviolet light and ethidium
bromide stain, nanogram levels of DNA can be observed. Ethidium bromide is a
mutagen, so other safer stains have been developed, which are often used in
teaching labs.
Most DNA is double stranded, but single stranded DNA and RNA can also be
separated by gel electrophoresis. Intrastrand base-pairing will cause anomalous
migration of the fragments, useful for studying polymorphisms.
Figure 1: A typical gel unit
Figure 2: A photo of an agarose gel
Reagents and Supplies:
DNA samples
Loading dye
Pipettes and tips
DNA Size standards
Electrophoresis Buffers
Gel stain
Agarose
Electrophoresis unit
Power supplies
Transilluminator
Cameras and/or digital recording dev