Section 6 Troubleshooting - Thermo Scientific A2-OK Operating And Maintenance Manual

Section 6
Troubleshooting
6-2 Horizontal System
Problem
Bands are not sharp, clear, and
even.
Samples are not moving as
expected through the gel, remain-
ing in the wells, or diffusing into
the gel.
When the comb is removed from
the gel, some sample wells are
ripped and damaged.
The gel seems to run slower
under the usual running conditions.
Solution
Always follow the proper procedure for preparing the agarose
product according to the manufacturers instructions. When
preparing the agarose, be sure all the agarose powder is in
solution before heating. In general, add powdered agarose to
distilled water and swirl to mix. Make sure all the powder is
equally wet to ensure proper melting. Heat in a microwave
oven, boiling water bath, or hot plate with occasional swirling
to melt and mix completely. Cool agarose liquid to below 60°
and cast. Note: Gel should be cast no thicker than 5mm to
avoid fuzzy banding. High percentage gels may thicken and
solidify rapidly and should be cast while still a liquid.
Check that a complete power circuit is achieved between the
unit and the power supply. Platinum wire and banana plugs
should be intact. To test, simply fill the unit with running buffer
and attach to the power supply without a gel or gel tray in the
unit. The platinum wires on both sides of the unit should pro-
duce small bubbles as the current passes through. If a complete
circuit does not exist, there will be few to no bubbles. Contact
Technical Services to schedule a repair.
Always make sure to allow the gel to solidify completely
before moving the gel tray, unit, or removing the comb. To avoid
damage to the sample wells, gently rock the comb back and
forth lightly to loosen, then slowly pull the comb straight up out
of the gel tray. This rocking helps to avoid suction as the comb
is removed. Alternatively, once casting is complete, simply sub-
merging the gel with running buffer will help loosen the comb.
Using a higher percentage of agarose that forms a tighter gel
matrix may remedy this problem as well.
The volume of running buffer used to submerge the gel should
only be between 3-5mm over the gel surface. Gel should be
completely submerged to avoid the gel from drying out, which
can smear the bands and possibly melt the gel(s) due to over-
heating. If excessive running buffer is added, the mobility of the
DNA decreases and band distortion may result. Excess buffer
causes heat to build up and buffer condensation inside the unit
may result.
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