How Do You Pack A Column In Preparation For Gel Filtration?


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Aun Jafery Profile
Aun Jafery answered
Here is the step by step procedure to packing a column in Gel Filtration Chromatography. First the glass column has to be suspended vertically in a level position on a ring stand. 1m tubing has to be connected to the stopcock and the stopcock has to be attached to the column. 50 mM Tris, pH 7.5 or Tris buffer has to be added next till about a third full. The G-75 or gel has to be swirled next, avoid stirring the same. Using a plastic funnel pour the gel in to the column. Open the stopcock and collect the eluted buffer after waiting for two minutes. Keep adding gel as the level of the liquid recedes. This should continue till the packed portion reaches near the yellow collar situated on the glass column. Leave a buffer of 1-2 cm close the stopcock. One m of tygon tubing should be connected at this stage with the lid of the column and the free end should be suspended in a 250 mL flask holding 200 mL of Tris buffer. Release the tubing after connecting it to the lid of the column. The 40 mL of buffer should be washed continuously and the flow rate noted.

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