Bio-Rad Sub-Cell® Model 192 Cell - Manual (Page 15)

3. Attach and tighten (10 lb.-in. torque) the straight fitting to the elbow-shaped fitting.
4. Connect tubing to the elbow-shaped fittings on the safety lid. Connect the other end of the
tubing to a suitable buffer recirculation pump (Section 6.3). Attach the tubing clips at all
tubing/fitting connections to insure that tubing does not disengage during electrophoresis.
5. Recirculate the buffer at a rate of 300-500 ml/min. Pumping at a higher rate will cause the
gel to float or slide off the tray causing variable sample migration rates during
electrophoresis. Check for any leaking in the fitting, tubing, and pump connections before
turning on the power supply and starting electrophoresis.
Note: If recirculation port fittings are to be removed, always cover the port holes by
replacing the port plugs (use 5 lb.-in. torque to tighten).
2.5 Electrophoresis
Once the agarose gel has solidified, sample loading and electrophoresis can begin. Agarose
gels can be run in many different types of electrophoresis buffers. Nucleic acid agarose gel
electrophoresis is usually conducted with either Tris-Acetate-EDTA (TAE) buffer or Tris-
Borate-EDTA (TBE) buffer. While TAE buffer provides faster electrophoretic migration of
linear DNA and better resolution of supercoiled DNA, TBE buffers have a stronger buffering
capacity and are less conductive than TAE buffers and therefore are used for longer or higher
voltage electrophoresis runs.
Note: Because of the higher voltages and resulting higher currents often used with the
Model 96 and Model 192, it is strongly recommended that only TBE buffers be used for
electrophoresis. TBE buffers have a stronger buffering capacity and are less conductive.
Thus, pH or temperature gradient formation during extended electrophoresis will be
reduced. If pH or temperature gradients cause uneven sample migration reduce the voltage,
add more buffer or recirculate the buffer during electrophoresis to eliminate these effects
(Section 2.4). Bio-Rad offers premixed 10x TBE buffers as well as individual buffer
reagents for use with the Sub-Cell systems (Section 6.3).
1. When placing the gel tray into the base, make sure that the sample wells are at the cathode
(black). DNA samples will migrate towards the anode (red) during electrophoresis.
2. Prepare the desired concentration of electrophoresis buffer (the electrophoresis buffer
used should be identical to the type used for gel preparation).
3. Submerge the gel under 4 to 6 mm of electrophoresis buffer. Do not fill buffer above the
max. buffer mark on the Sub-Cell base.
4. Prepare samples for gel loading. The maximum sample loading volume for Bio-Rad
combs is listed in Section 6.2. Loading volume is dependent upon the type of comb used
(i.e., well thickness and length) and thickness of the gel.
5. Once loading volume is determined, samples are made dense for underlaying into sample
wells by using standard nucleic acid sample loading dyes (refer to Section 3.4 for sample
loading dye preparation). Add loading dye to a final 1x concentration.
6. Load the samples into the wells using standard pipets or multichannel pipets.
Note: Sample wells are often difficult to see. Well visualization can be enhanced by placing
black paper or tape under the base or tray where comb placement or well formation is
common.
7. Place the lid on the DNA cell carefully. Do not disturb the samples. The Sub-Cell systems
lid attaches to the base in one orientation only. To attach the lid correctly, match the red
and black banana jacks on the lid with the red and black banana plugs of the base.
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