Bio-Rad Sequi-Gen GT Sequencing Cell - Manual (Page 21)

3. To avoid buffer spills and cell tipping accidents, adjust the leveling screws on the universal
base, as necessary.
• To insure that the unit will not tip over during electrophoresis, make sure the leveling
feet threaded rods are at least 1 cm deep into the threaded boss of the base.
• At this time, test whether the IPC assembly is properly aligned in the universal base by
attaching the top and bottom safety covers. The IPC assembly may have to be shifted
to the right or the left to properly attach the safety covers. After this final alignment is
complete, remove the safety covers.
4. Fill the upper buffer chamber (the IPC) with running buffer (1x TBE) using the flared
portion of the panel as a fill spout.
• The level of the buffer should be about 1 cm from the top of the fill spout at all times
during the run.
• Remove the comb(s) from between the glass plates.
• Thoroughly rinse the resulting well(s) or gel front using a syringe with a needle,
or disposable plastic transfer pipet (catalog number 223-9911).
• If using a sharkstooth comb, insert the comb with the teeth facing the gel front. Lower
the comb toward the gel surface until the teeth of the comb just touch the gel surface.
5. Fill the lower buffer chamber with 350-500 ml of the running buffer. Refer to Appendix
7.1 for running buffer recipes.
Caution: Do not fill the lower chamber with more than 500 ml of buffer. The lower buffer
chamber holds the entire volume of the upper buffer chamber should a leak develop in the
IPC. Buffer levels over 500 ml will not allow the entire volume of the upper buffer chamber
to be contained in the universal base.
6. Attach the top and bottom safety covers and pre-electrophorese the gel at normal operating
voltage or power (see Section 4.7), if desired, to increase the gel temperature.
• Pre-electrophoresis prior to sample loading will create a uniform gel temperature and
bring the gel temperature to the recommended run temperature. This will help eliminate
any smile patterns from developing early in the run.
Note: Gel electrophoresis buffers can be heated to 50 ˚C in a microwave before adding
buffer into the upper buffer chamber. This will reduce the time needed to bring the gel to
the appropriate run temperature before sample loading, and will greatly reduce
pre-electrophoresis time.
Warning: The upper buffer level may drop slightly due to evaporation as the system
becomes warmer. Make sure that the upper chamber is always filled with buffer during
electrophoresis. Do not allow the buffer level to drop below the level of the notched
(shorter) IPC glass plate at any time during electrophoresis, as this may cause arcing and
cell damage. Additionally, never allow the gel to exceed 60 °C under any circumstance.
This excessive heat may crack the plates or cause the IPC/glass bond to deteriorate.
4.5 Loading the Gel
1. Turn off the power supply, and remove the top safety cover.
• Rinse the well(s) with a syringe with needle, or disposable plastic transfer pipet
(catalog number 223-9911), (to remove urea) before applying the samples to the gel.
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