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Star Republic: Guide for Biologists

Sephadex column purification of DNA probes

1. Sephadex column can be used to separate DNA from unincorporated nucleotides. When working with radiolabeled probes, proper protection and waste disposal are required.

2. Swell the Sephadex (G-50-80) in water for several hours.

3. Remove excess water, add equal volume TE. Mix, allow Sephadex to settle down, remove supernatant. Repeat several times.

4. Store the Sephadex in TE and 0.1% sodium azide at room temperature or autoclave to sterilze and store at room temperature.

5. Plug the bottom of a 1 ml syringe with siliconized glass wool or use a 3 ml chromatogrphy column.

6. Fill the syringe or column with Sephadex G50 using a pasture pipette.

7. Place the Sephadex-filled syringe into a 15 ml Corex tube and centrifuge at 1,500 rpm for 2 minutes to pack down the Sephadex.

8. Repeat Step 6 and 7 until the syringe or column is 80-90% full.

9. Wash twice the Sephadex column with TE. Spin and dump out flow-through.

10. Put a 1.5 ml eppendorf tube at the bottom of the Corex tube to collect the sample. Place the bottom of the Sephadex column in the center of the eppendorf tube.

11. Add labeled sample to the Sephadex column, centrifuge at 1,500 rmp for 2 minutes. Labeled probe will be in the flow-through.

12. The speed and time of centrifugation may vary depending on the centrifuge used.