Part A: The column has been mounted on a ring stand. 50mL of Tris buffer has been added while the outlet is closed. Now the column is filled with a slurry of Sephadex G-75 (note that it must be stirred just before pouring into the column).

Part B: The sephadex has settled and the outlet is taped just below the top of the column and opened so buffer will flow through the column. Notice that the effluent drips from the outlet slowly. During part C, the outlet is lowered to slow the dripping.

Part C: More slurry is added frequently, keeping the top surface of the gel 1-2 cm below that of the column (our sample column is lower to make it more visible). Notice a distinct difference between packed material and unpacked slurry. Video shots were taken at several points during the packing.

Part D: The top of the column is closed and the Mariotte flask above the column is connected to the top of the column through tubing. The column is washed with about 300 mL of buffer. 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.

Part E: 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.

Part F: 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!

Part G: The sample is drawn into the gel bed. Notice the height of the liquid decreases during this part.

Not shown: 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.