An inexpensive alternative to glassmilk for DNA purification

 

 

<center> <h3> An inexpensive alternative to glassmilk for DNA purification</h3> </center>

 

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 C.

 

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 C for 5-10 min with occasional mixing.

3. Add 10 ul of the silica suspension. Vortex gently. Stand for 5 min at room temperature with occasional mixing. One mg of the silica (=10 ul of the silica suspension) binds 3 -4.5 ug 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 500 ul 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.