Hoefer HE99X User Manual

Page 15

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Preparing for electrophoresis

Optional: To monitor separation progress, either add
0.5 µg/ml (final conc.) of ethidium bromide to the
running buffer now or add 50 µg/ml (final conc.)
ethidium bromide to the sample buffer. To visualize
progress, turn off the power supply, remove the lid
assembly, and hold a portable UV lamp near the gel.
Note: Adding ethidium bromide to the running or
sample buffer slows migration slightly. Detection by
this method is not as sensitive as by staining after the
electrophoresis run. See the DNA detection section,
for more details (page 14).

Fill the chamber with buffer until the gel is
submerged ~

~1 mm.

Load the samples. Add the sample to 1/5 volume of
the sample loading buffer. Mix each sample and load
into a well with a micro-pipet, taking care to avoid
puncturing the well bottom or entrapping bubbles.

Place the lid on the unit so that the cathode (black
lead) is at the end nearest the samples. (Nucleic acid
samples migrate toward the anode.)

Connect the color-coded leads (red to red, and black
to black) to an approved power supply. Set the voltage
and timer (if available). Agarose gels are typically
run at constant voltage under a voltage gradient in
the range of 2–5 V/cm. The distance between the
electrodes is ~

~26 cm, so a setting of 130 V results in

a gradient of 5 V/cm.

Caution! Wear UV safety goggles
and protect skin when using a
UV lamp.

Note: Refer to the Buffers,
volumes, and notes section
for additional information and
guidelines on page 10.
See page 12 for a sample
loading buffer recipe and well
volumes for various comb sizes
in gels of different thicknesses.

Important! If running two sets
of samples in one gel, monitor
the run closely and stop
electrophoresis when the marker
dye approaches the wells in
the center.