G50 Sephadex spin column

MATERIALS

G50 Sephadex (DNA grade F; GE Healthcare)

Tris-EDTA (10 mM), DEPC-treated

Centrifuge (swinging bucket)

Culture tubes, 14-mL round-bottom

Falcon tube (50-mL), for use if large buckets are available (see Step 6)

Glass wool, RNase-free (baked)

Pasteur pipette

Syringe, 1-cc disposable

To prepare the spin column:

1. Hydrate Sephadex G50 beads in DEPC-treated 10 mM Tris-EDTA.
2. Remove the plunger and cap from a disposable 1-cc syringe (save the cap).
3. Plug the bottom of the syringe barrel with RNase-free glass wool (“fluff” from the back of a sterile disposable pipette also works if glass wool is not available).
4. Back-fill the syringe with G50 Sephadex using a Pasteur pipette.
5. Hang the syringe in a disposable 14-mL round-bottom culture tube.
6. Centrifuge briefly at 600 rpm in a swinging bucket centrifuge to spin down the matrix. Use Setting 2 on a standard benchtop blood centrifuge. If large buckets are available, use a 50- mL Falcon tube as an improvised tube adapter.
7. Add ~400 μL of 10 mM Tris-EDTA to the top of the syringe, and centrifuge at 600 rpm for 4 min to pack the matrix.
8. Remove syringe and discard flowthrough liquid in the culture tube. Replace the cap on the syringe and put it back into the culture tube.