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66 Bartlett
Table 1
Recommended Agarose Concentrations
for DNA Electrophoresis
Percentage agarose (w/v)
Molecular weight range
0.6% 1000 –20,000 bp
long PCR
1.0% 1500 –5000 bp
2% 1100 –2000 bp
3% 1110 –500 bp
nucleotides, although silver staining may be applied to these gels the fragility of the
gel often precludes this.
Agarose gels, however, are more robust and easy to prepare. Although resolution is
poorer, some modern forms of agarose claim separations that rival that of acrylamide
gels. The major strength of agarose-based gels is the greater range of separation.
Conventional agarose electrophoresis can separate DNAs from 200 to 50,000 bp which
is more than adequate for PCR-based systems. Adaptations of agarose electrophoresis,
for example, those using pulsed electric fields, can be used to separate DNA fragments
of up to 10 Mbp.
2.1. Selecting Conditions for Agarose Gel Electrophoresis
Those interested in understanding the electrophysical properties governing migration
of DNA in agarose supports can refer to a number of molecular biology texts that
cover these areas or alternatively visit the web (1) for a useful guide to electrophoresis
with agarose gels.
2.1.1. Agarose Concentrations
Table 1 outlines recommended agarose concentrations for gel electrophoresis of
DNA. Note that modern specialist formulations of agarose may require alterations to
this table (e.g., Nusieve, etc.).
2.1.2. Buffers
Two buffering systems are commonly used for agarose gel electrophoresis of DNA;
of these, Tris acetate EDTA buffer (TAE) is more widely accepted because it facilitates
recovery of material from agarose. However, it has a relatively low buffering capacity,
and recirculation of buffer may be required over long electrophoresis runs (4–6 h).
Tris borate EDTA (TBE) is preferred for small molecules and longer electrophoresis
times because of its higher buffering capacity. We have used both with good results.
Independent of the buffer selected, attention should be paid to the depth of buffer. In
most systems 3 to 5 mm of buffer should cover the gel. Insufficient buffer may allow the
gel to dry out during the run, whereas excessive buffer will reduce the current through
the agarose support, promote heating and decrease DNA motility. Electrophoresis is
performed applying a voltage of between 1 to 5 V/cm (where cm is the distance in
centimeters between the electrodes in the gel tank).