C.B.S. Scientific DCX-700 - Manual (Page 17)

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4. Insert blotting casettes into core making sure
that red side faces outward. See diagram 5.
5. Close doors and re-latch by pressing down on
the white latches so that assembly looks like
that shown in figure 6. If running one blot, slide
white reservoir conversion plate into the side
without the blotting cassette.
5.2 Electro- Blotting Procedure
1. Place stirring bar in bottom corral of lower res-
ervoir. Place frozen freezer blocks in side recep-
tacles. Place core/blotting cassette assembly
into lower reservoir. The anode (red) and cath-
ode (black) electrodes are color-coded on both
the core/cassette assembly and lower reservoir.
Ensure the red dot on the cassette assembly is
on the same side as the red receptacle on the
lower reservoir.
2. Pour 1 liter of freshly prepared, chilled (4º) buffer
into lower buffer reservoir. Buffer will percolate
into central core.
3. Attach safety cover. The unit should look as
shown in figure 7 and is ready for power.
4. Connect the leads to the power supply, match-
ing the color-coded red to red and black to
black.
See Section 6.2 for recommended
power conditions. Begin transfer by electro-
phoresis.
5.3 Removing the Blot
1. Turn the power supply off and disconnect the
leads from the power supply. Remove the
safety cover from the unit, by placing thumbs
on white posts next to red & black connectors,
then pushing down while pulling up with fingers
under lid
.
Do not remove safety cover by pulling up on
leads!
2. Blotting cassettes can be removed by leaving
the core in place and opening the top latches of
the core, opening the doors and lifting the cas-
settes out. Unlatch the blotting cassettes and
remove blot from blotting sandwich.
SECTION 6
Running Conditions
6.1 Recommended Power for Slab Gels:
Precise electrophoresis conditions will vary according to the number and type of
gels used, buffer conditions employed, power input, and the general goal of the
experiment. Refer to reference section 6.3 for in depth discussions on practical and
theoretical approaches to protein gel electrophoresis.
6.1.1 ClearPAGE Gels
Run Voltage Starting Current Ending Current Approx. Run TIme
180VDC 90mA/gel 40mA/gel 30-75 minutes
6.1.2 General Recommendations
If running only one gel, keep the volts the same but reduce the mA’s by half. Keep in
mind that as the thickness of gel increases, the mA’s increase proportionally.
At constant voltage, the proteins will migrate at a constant rate during electropho-
resis with adequate heating appropriate for denaturing gels. Increasing the voltage/
mA (for a single gel thickness and percentage) will speed mobility but increase the
risk of overheating.
If using freezer blocks, the power input and the migration rate can be increased.
The joule heating generated by the higher power is offset by the cooling effect of
the buffer between the gels. Exact conditions should be determined empirically. We
recommend using at least one freezer block for 2 reasons; less buffer usage and
cooler buffer temperature. If using both freezer blocks, outside lanes can still be
viewed through the corners of the tank. If it is important ro view the entire gel during
electrophoresis, use only 1 freezer block and place it at the back of the tank.
6 1 3 Tris-Glycine Gels
For SDS-PAGE Tris-Glycine (Laemmli) buffer systems with two 1.0mm thick gels at
room temperature use the following conditions at constant voltage:
80VDC until samples have fully entered stacking gel
120VDC @ 60mA-90mA/gel (depending on gel type) thereafter until dye is
near bottom of gel