Hoefer HE33 - Manual (Page 23)

• p15
DNA detection
DNA can be detected either by the fluorescence of
bound ethidium bromide or by autoradiography of
radiolabeled DNA.
Ethidium bromide (0.5 µg/ml) can be added to
running buffer to monitor sample progress because
the dye’s fluorescence under a UV lamp reveals
band location. (To check progress, 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.) View the sample under
366-nm UV light or reduced intensity 302-nm UV
light to reduce photonicking.
To reduce the background fluorescence of unbound
ethidium bromide, the gel can be destained by
soaking it for 5 minutes in 0.01 m MgCl
2
, or for 1
hour in 0.001 M MgSO
4
. Destaining makes it easier
to detect small quantities (less than 10 ng) of DNA.
(Sambrook, section 6.15).
Bibliography
Ausubel, et al., (eds). Current Protocols in Molecular
Biology. Greene Publishing and Wiley-Interscience.
New York (1993).
Sambrook, J., Fritsch, E.F., and Maniatis, T.,
Molecular Cloning: A Laboratory Manual. Cold
Spring Harbor Laboratory Press (1989).
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 about 15%.
Note: Minimize the staining time
to prevent small nucleic acid
fragments from diffusing out of
the gel.