Hoefer SUB Series - Manual (Page 27)

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p19
Agarose Gel Electrophoresis
DNA Mobility
DNA fragments as small as 1 kb or less can be
separated using agarose gel electrophoresis. For
fragments smaller than 0.1 kb, polyacrylamide gels
are more suited.
RNA Mobility
Either before or during electrophoresis, RNA should
be denatured. For example,
• RNA fragments which have denatured with glyoxal
and dimethyl sulphoxide can be separated on
neutral agarose gels, or
• RNA can be fractionated on agarose gels containing
methylmercuric hydroxide or formaldehyde.
RNA samples usually require longer runs or buffers
that are easily depleted, so it is necessary to circulate
the buffer. Northern analyses should not normally be
run on a mini gel tank.
Separation Performance
Gel concentration, running buffer, voltage,
temperature, conformation, and the presence of
ethidium bromide all affect separation results.
Gel Concentration Selection
The range of fragment sizes to be separated will
determine the choice of agarose concentration for
a gel. Typical agarose concentration is 0.5% to
3.0%. For large DNA fragments low-percentage
gels are required, while for small DNA fragments,
high-percentage gels are recommended. Weak gels
(< 0.5% agarose) should be electrophoresed at low
temperatures (e.g. ~ 4 °C). Agarose gels of 0.75% to
1.0%, for routine electrophoresis, are recommended
for a wide range of separations (0.15 to 15 kb).
2 – 4% agarose gels are usually selected for PCR
fragment resolution.
If the gel has to be subsequently photographed, thin
gels (2 – 3 mm) with low-percentage agarose are better
than thick or high-percentage gels. The latter produce
increased opaqueness and autofluorescence.