Hoefer HE99X - Manual (Page 22)

RNA mobility
RNA can also be separated on the basis of
size. To avoid irregularities due to secondary
structure, RNA is denatured either before or
during electrophoresis. For example, RNA
fragments previously denatured with glyoxal
and dimethylsulfoxide 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, and so require
circulation. The Hoefer SUB20C and SUB25C
horizontal units are recommended for this appli-
cation rather than the HE33.
DNA detection
DNA can be detected either by the fluorescence
of bound ethidium bromide or by autoradiogra-
phy of radio-labeled DNA.
Ethidium bromide (0.5 µg/ml) can be added
to running buffer to monitor sample progress
because the dye’s fluorescence reveals DNA
under UV light. (To check band location, turn
off the power supply and remove the lid of the
agarose unit. Hold a portable UV lamp near the
running tray. Replace the lid and turn on the
power again to resume electrophoresis.)
Alternatively, after electrophoresis, stain the
gel in an ethidium bromide solution (0.5 µg/
ml H
2
O) for 15 to 60 minutes and then
view or photograph the sample on a UV
transilluminator.
To photograph the gel, either place the running
tray on the transilluminator surface or slide the
gel onto the surface for maximum exposure. The
running tray is 95% transparent to 302 nm light
and 40% transparent to 254 nm light. If you
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Caution! Ethidium bromide is
a known mutagen. Always wear
gloves when handling.
Caution! Wear UV safety goggles
and protect skin when using any
UV light source.
Note: Ethidium bromide slows
DNA migration by ~
~15%.
Note: Minimize the staining time
to prevent small nucleic acid
fragments from diffusing out of
the gel.
Note: For an example of
RNA electrophoresis, refer to
Molecular Cloning: A Laboratory
Manual by J. Sambrook and
D.W. Russell.