C.B.S. Scientific DASG-250-02 User Manual
Page 15
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15
Adjustable Slab Gel Manual 07/13
SECTION 4
Running Conditions
4.1
Recommended Power
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 the reference
section for in depth discussions on practical and theoretical approaches to protein gel
electrophoresis.
Using standard SDS-PAGE buffer systems apply 1-10 VDC/cm of gel. For sequencing
applications use 50 VDC/cm. If running two gels in the Dual Units, keep the volts the same but
double the mA. It is also true that if the thickness of gel increases, increase the mA
proportionally.
At constant voltage, the proteins will migrate at a constant rate during electrophoresis 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.
The sample migration rate can be increased by raising the input power. This can be done son
systems which employ “active” temperature control such as Dual Slab Gel Units and Dual Mini-
Vertical Gel Units. The joule heating generated by the higher input power is offset by the cooling
effect of the water jacket between the gels. Exact conditions should be determined empirically
but could be increased at least in the 20% range.
4.2
Recommended Buffers and Reagents
Pre-mixed acrylamide stock solutions are the method of choice. Use according to manufacturer’s
instructions.
Typical ‘scratch’ recipe for a 4% acrylamide gel:
10mls 40% acrylamide
6.6mls 2% Bis-acrylamide stock
5mls 10X TBE
78.4mls dH
2
0
750ul 10% APS
50ul TEMED
1. Make up 0.5X TBE buffer
2. After gel apparatus is set-up and ready for the gel to be poured, add 750ul
fresh 10% APS solution to the acrylamide solution.
3. Add 5 to 10 ul TEMED and using a 10 to 25ml pipette, quickly “pour” the gel.
4. Allow the gel to polymerize at least 60 minutes.
5. Remove the comb after polymerization and wash out wells with 0.5X TBE
(acrylamide will seep into the wells).
6. Fill upper and lower chambers with 0.5X TBE
7. Pre-electrophorese gel, if needed, 20-30 minutes.
8. Load wells with samples.
9. Monitor migration with dye markers.