Bio-Rad Bio-Gel A Agarose Gel - Manual (Page 7)

9. After equilibration with the starting buffer, the column
is ready for sample application.
Section 4.2 Column Operation
1. Equilibrate the column you will be working with by
pumping 5–10 column volumes of run buffer over the
column at the flow rate at which the separation will be
run. Typically slower flow rates, and taller columns,
provide superior separations.
2. Apply your sample as close to the column inlet as
possible.
3. Follow sample application with run buffer. Do not
allow the column to run dry.
4. Monitoring your chart recorder, collect the
components as desired.
Section 4.3 Void Volume Determination and
Calibration
The void volume (V
o
) of the bed should be
determined and the bed tested for uniformity of eluant
flow before applying experimental sample. To assure
accurate V
o
values, test substances must exhibit minimal
interaction with the gel medium and should be large
enough to assure elution in the true void volume. Blue
9
be trapped in the bed support or the column end piece.
Close the exit stopcock.
4. Fit the column with a reservoir and filling funnel and
add the gel slurry. Allow the gel to settle until it
reaches a 3–5 cm bed height.
5. Open the stopcock and allow buffer to flow until a
stable gel bed has packed.
6. Connect the flow adaptor tubing to the pump, fill it
with buffer, and make sure it is free from air bubbles.
Close the exit stopcock, remove the excess buffer from
the reservoir, and attach the flow adaptor to the column.
(Inserting it at a slight angle makes it easier to avoid
trapping air bubbles.)
7. Open the column outlet and pump at least 5 bed
volumes of run buffer through the column at a flow
rate which is 10–20% higher than the flow rate at
which the actual separation will be run. (For typical
flow rates see Table 2.)
8. Switch off the pump and close the column outlet.
Adjust the flow adaptor until it is in contact with the
gel surface. During this step, you may have to
disconnect the flow adaptor tubing from the pump to
allow buffer to flow back through the tubing as you
bring the flow adaptor in contact with the gel bed.
8
4006139A.qxd 6/23/98 8:11 AM Page 8