Bio-Rad ReadySub-Cell GT Cells - Manual (Page 12)

4. Allow 20—40 min for the gel to solidify at room temperature.
5. Carefully remove the comb from the solidified gel.
6. Remove the tape from the edges of the gel tray.
7. Place the tray onto the leveled Sub-Cell base so that the sample wells are near the cathode (black).
DNA samples will migrate toward the anode (red) during electrophoresis.
8. Submerge the gel beneath 2 to 6 mm of 1x electrophoresis buffer (see Section 3, Gel and
Electrophoresis Reagent Preparation). Use greater depth overlay (more buffer) with increasing voltages
to avoid pH and heat effects.
2.3 Electrophoresis
After 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 buffers
provide faster electrophoretic migration of linear DNA and better resolution of supercoiled DNA, TBE buffers
have a stronger buffering capacity for longer or higher voltage electrophoresis runs. Bio-Rad offers premixed
50x TAE and 10x TBE buffers, as well as individual buffer reagents for use with the Sub-Cell GT systems.
1. Prepare samples for gel loading. The maximum sample loading volumes for Bio-Rad’s combs are 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.
2. When loading volume is determined, add standard nucleic acid sample loading dye to a final 1x
concentration to make samples dense for underlaying into sample wells (see Section 3, Gel and
Electrophoresis Reagent Preparation, for sample loading dye preparation).
3. Load the samples into the wells using standard pipets. Multichannel pipets can only be used for loading
samples with the Bio-Rad MP combs (see Section 6.2).
Note: Sample wells are often difficult to see. Well visualization can be enhanced by placing black paper or tape
under the base or trays in the are of comb placement and well formation.
4. Place the lid on the DNA cell carefully. Do not disturb the samples. The Sub-Cell GT system lids attach to the
base in only one orientation. 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.
5. Power requirements vary depending on gel thickness, length, agarose, concentration, and type of
electrophoresis buffer used. Refer to Tables 2.3 and 2.4 below for relative sample migration rates for the
different Sub-Cell GT systems and for DNA size migration with sample loading dyes.
Note: Buffer recirculation is not required for most standard DNA and RNA agarose gel electrophoresis.
If buffer recirculation is required, simply turn off the power supply, remove the safety lid, and mix the
running buffer as desired. After the buffer has been mixed, reconnect the safety lid and continue with
electrophoresis.
Table 2.3 Relative sample migration rates*
Bromophenol Blue
Cell Type Voltage Migration Rate
Sub-Cell GT cell, 15 x 15 cm gel 75 V 3.0 cm/hr
Wide Mini-Sub cell GT, 15 x 10 cm gel 75 V 4.5 cm/hr
Mini-Sub cell GT, 7 x 10 cm gel 75 V 4.5 cm/hr
* These sample migration rates were determined based on a 0.5 cm thick 1.0% agarose gel using Bio-Rad’s
Molecular Biology Certified Agarose in 1x TAE electrophoresis buffer (diluted from Bio-Rad’s Premixed 50x
TAE Buffer). Migration rates will vary depending on the voltage, current, and type of agarose or buffer used.
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