Preparation of Silica

1. Suspend 5 g of silica (Sigma, S-5631) in 50 ml of PBS.

2. Allow the silica to settle for 2h.

3. Discard the supernatant containing fine particulate matter.

4. Repeat steps 2 and 3.

5. Spin for 2 min at 2000 g. Discard the supernatant.

6. Add 3 M NaI to make a final concentration of 100 mg silica /ml.

7. Store the silica suspension in the dark at 4℃.

Recovery of DNA from Agarose Gel Using the Silica Suspension.

1. To the agarose gel containing DNA fragments, add two volumes of 6 M NaI.

2. Incubate the agarose in 6 M NaI at 55℃ for 5-10 min with occasional mixing.

3. Add 10μl of the silica suspension. Vortex gently. Stand for 5 min at room temperature with occasional mixing. One mg of the silica (=10μl of the silica suspension) binds 3 -4.5μl of DNA.

4. Spin for 1 min in a microcentrifuge. Discard the supernatant.

5. Pulse spin, and carefully remove residual liquid.

6. Suspend the pellet in 500μl of 50 mM NaCl, 10 mM TrisHCl pH 7.5, 2.5 mM EDTA, 50%(v/v) ethanol.

7. Spin for 1 min. Discard the supernatant.

8. Repeat steps 6 and 7.

9. Allow the pellet to air-dry for 10 min.

10. Add an appropriate volume (at least one pellet volume) of TE. Vortex gently to resuspend the pellet, and stand for 5 min at room temperature with periodic agitation.

11. Spin for 1 min. Transfer the supernatant into a new microfuge tube.