The Department of Biological Sciences at the University of Alberta brings you this video tutorial specifically relevant to your student laboratory courses, specifically microbiology. If you're a student at any school of biology, this information will be helpful for learning how to use Sephadex gel filtration for chromatography in the lab.
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.
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.
Part C: More slurry is added frequently, keeping the top surface of the gel 1-2 cm below that of the column. Notice a distinct difference between packed material and unpacked slurry.
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.
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.
Part G: The sample is drawn into the gel bed.
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 attached to an automatic fraction collector. The column should be run slowly and should be set up so it does NOT dry out.
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