Microbiology 345: Setting up a gel filtration column
The column has been mounted on
a ring stand and the outlet closed.
50mL Tris buffer is added to the
column.
The column is filled with a slurry of 50% sephadex G-75. The slurry must be stirred thoroughly before pouring.
More sephadex is added frequently to pack the column. The final surface of the gel bed should be 1-2 cm
below the top of the column (note that ours is lower so the top of the gel bed is visible). Notice a distinct
difference between packed material and unpacked slurry. Video shots were taken at several points during
the packing. The outlet is lowered throughout the packing procedure to slow the rate of dripping.
The outflow is closed and the Mariotte flask
disconnected. The cap is taken off and the buffer
above the gel is removed with a pipette.
Once the sephadex has settled, the outlet is taped just
below the top of the column and opened to allow buffer to
flow through the column.
The top of the column is closed tightly. The Mariotte flask is attached
through the tubing to the top of the column. The column is washed with
300 mL buffer.
The sample to be run is gently loaded by using a pasteur pipette. The tip of the pipette is held
against the wall of the column and moved in a circular motion so the sample slowly layers
onto the gel bed. Do NOT break the pasteur pipette!
The effluent is
collected in a beaker.
Once the wash is complete the column set-up is taken to the cold
room and run overnight to equilibrate. Note: it is vital that that the
column be set up properly so it does not dry out, especially if left
unattended. The pressure head (space between the buffer and
outlet) should never be more than 20 cm.
The sample is drawn into the gel bed. Notice the
height of the liquid decreases during this part.
The column is
washed 2 -3 times
with 2 mL aliquots of
buffer. Once washed,
the outlet is closed
and the column is
filled with buffer.
The Mariotte flask
with 400-500 mL
buffer is attached. A
pressure head of 15
cm is used. The
outlet is atteched to
an automatic
fraction collector.
The column should
be run slowly and
should be set up so
it does NOT dry out.