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Star Republic: Guide for Biologists

Purification of DNA from agarose gels (silica beads)

Agarose gel is dissolved by NaI and DNA purified with glassmilk or silica beads.

1. Excise DNA band from agarose gel. Place in one or more eppendorf tubes, filling less than one third of the tube.

2. Add 2-3 volumes 6 M NaI.

3. Incubate the agarose at 55 C for 5 minutes or until the gel slice is completely dissolved.

4. Add 5-10 ml of glassmilk or silica beads.

5. Mix by inverting the tubes several times.

6. Spin for 1 minute in a microcentrifuge, remove the supernatant.

7. Resuspend the pellet in 500 ml of Wash Buffer (50 mM NaCl, 10 mM Tris-HCl pH7.5, 2.5 mM EDTA, 50% (v/v) ethanol).

8. Centrifuge for 1 minute and remove the supernatant.

9. Repeat the wash with the Wash Buffer twice.

10. Air dry the pellet.

11. Elute DNA from glassmilk/beads with 10-30 ml of TE or water. Stand at room temperature for 5 minutes. For large fragment, incubate at 55 C for 5 minutes.

12. Centrifuge 30 seconds and carefully remove supernatant containing DNA to a fresh tube.

13. Elute the second time with a smaller volume of TE or water.

Preparation of silica beads

1. Weight 1 gram of silica beads.

2. Add 10 ml PBS, allow the silica to settle (1-2 hours), then remove the supernatant.

3. Repeat step 2 once.

4. Add 10 ml PBS, pellet the beads by centrifuging at 2,000 g for 2 minutes. Remove the supernatant.

5. Add 10 ml 3 M NaI. Final concentration of silica beads ~100 mg/ml.

6. Store the silica suspension in the dark at 4 C.