Blotting Applications; Required Steps For Proper Blotting Results; Section 3 Blotting Applications - BIO RAD 200/2.0 Instruction Manual

3. Press LIMIT to view the set current limit. The red indicator light above the button should
be on, and the set limit will be shown on the LED display. To adjust the current limit,
follow the procedure in Section 2.1.
4. Press TIMER to view the remaining time on the timer. The red indicator light above the
button should be on, and the time remaining will be shown on the LED display. To change
the time remaining for the run, follow the steps detailed in Section 2.2, item 2.
5. To view the resistance laod, power load, and volt-hours settings, press AMPS, and then
press either RAISE or LOWER. The indicator light above AMPS will go off, and the
LED display will alternately flash rLD and the resistance value in thousands of ohms.
Press RAISE or LOWER again, and the LED display will alternately flash PLD and the
power output in watts. Press RAISE or LOWER again, and the LED display will alternately
flash E-H and the volt-hour reading in thousands of volt-hours.
Section 3

Blotting Applications

3.1 Required Steps for Proper Blotting Results

Observe the following guidelines to avoid mishaps that may result in serious damage to
the instrument or injury to the operator:
1. Read the Trans-Blot
2. Do not attempt high intensity transfers or overnight runs until you are completely familiar
with your system.
3. Pre-equilibrate electrophoresis gels in transfer buffer before beginning transfer to remove
contaminating electrophoresis buffer salts or neutralization salts that will increase the
conductivity of the transfer buffer. The length of time required for pre-equilibration
depends on the gel thickness, and can range from 20 minutes for 0.75 mm thick SDS
polyacrylamide gels to 1 hour for 3-6 mm thick DNA agarose gels. The gel can be
pre-equilibrated in a shorter time if the pre-equilibration buffer is changed several times.
4. Use the General Guide to Transfer Buffers and Running Conditions (Table 1) as a guide-
line in setting current limits on the power supply. Unless proper current limits have been
set, uncontrolled conductivity changes may result in full power being delivered to the
Trans-Blot cell. This is a safety hazard due to the excessive heat (power). In this situation,
the transfer buffer may boil and evaporate, causing the gel holder and electrode cards to
deform.
5. Operating the Trans-Blot cell in the coldroom is an inadequate means of controlling transfer
buffer temperature. For high power applications, efficient heat removal is obtained only
by using the Super Cooling Coil connected to a refrigerated recirculating bath capable
of 200-300 watt heat extraction at ambient evaporation temperature.
6. Place a stirring bar inside the Trans-Blot cell and stir during transfer to help maintain uniform
buffer conductivity and temperature.
7. Buffer preparation is extremely important. Do not pH adjust transfer buffers unless specifically
indicated in the instruction manual. Use only high quality, reagent grade methanol. Do not
reuse transfer buffers.
® cell instruction manual before beginning electrophoretic transfers.
6