Protocols

Spin columns preparation and use

Prepare column.

1. Prepare Sephadex.
   Hydrate Sephadex by incubating 50 g Sephadex G-25 in 400 mL buffer* at room temperature for 3 h or heated in water bath at 90°C for 1 h. (Do not stir)
2. Roll up glass wool and stuff into 1 mL plastic syringe. The amount of glass wool should fit into 0.1 mL
3. Place the syringe in a 13 x 100 mm test tube.
4. Pipet the slurry of Sephadex into the column and let drain.
5. After the gel settles, add more.
6. Centrifuge for two minutes.
7. Add more Sephadex and centrifuge for 10 min.

De-salting

1. The buffer can be exchanged by eluting a total of 2 mL through column.
2. Centrifuge for 10 min
3. Put microcentrifuge tube in bottom of a larger test tube.
4. Place the syringe on top so that the eluant will flow into the microcentrifuge tube.
5. Add 100 mL of sample. If sample is less than 100 mL, first load the sample and then add additional buffer to add a total of 100 mL to the column.

Test spin column and centrifuge speed

1. Prepare two solutions (1) 50 mg/mL Blue Dextran and (2) 50 mg/mL bromophenol blue in water.
2. Load 100 L each solution on top of spin column
3. Centrifuge 10 min.
4. The Blue Dextran should all come through
5. Bromophenol blue should stay in the column
    

* This buffer will be the buffer that the desired molecule will be dissolved in after the procedure.

Typically this would be 50 mM Tris-HCl (pH 8.0), 1 mM EDTA