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. |
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