Sephadex G-50 spun column purification

 

Sephadex G-50 spun column purification

Spin column purification can be used to change buffers without a concomitant change in solution volume, to remove protein contaminants or to purify plasmid DNA suitable for sequencing.

1) Plug a 1ml syringe barrel with siliconized glass wool and fill to the top with a sterile suspension of Sephadex G50 equilibrated with H₂ O.

2) Once filled place the syringe barrel into a 10ml centrifuge tube and spin at 3,000g for 5 minutes in a MSE Centaur with swingout rotor.

3) After centrifugation, 100-200ul of the required buffer is added to the top of the column and the column re-centrifuged. Repeat 3 times.

4) The conical base of a sterile microfuge tube is placed into the bottom of the emptied 10ml centrifuge tube and the syringe barrel replaced. Apply DNA sample to the top of the column in 100-200ul of the appropriate buffer and centrifuge as before. Eluate collected in the microfuge tube contains DNA for further manipulation.

If plasmid DNA is required for sequencing purposes, the column should be set up as described, but Sephacryl S-400 is substituted for Sephadex G50.