Plasmid using Silica Gel

Silicon dioxide

Suspend 60 g Silica particles (silicon dioxide, SiO2; Sigma Chemical Co., St. Louis, Mo.) in 500 ml ddH2O water in a total volume of 500 ml for 24 hours at room temperature.

Remove supernatant by suction

Add 500 ml ddH2O, resuspend silica by vigorous shaking.

After 5 h at room temperature remove 440 ml of the supernatant by suction

Add 600 ul of HCl (32%, wt/vol) to adjust the suspension to pH 2.

The resulting suspension of silica coarse (SC) is sufficient for approximately 1,500 NA purifications.

Transfer 4 ml into glass bottle and autoclave for 20 min at 121°C to destroy any contaminating NA.

SC is stable for at least 6 months when stored at room temperature in the dark.

Celite

Add 50 ml of H20 and 500 ul of 32% (wt/vol) HCl to 10 g of high-purity, analytical grade Celite obtained from Janssen Chimica (Beerse, Belgium).

Lysis buffer L6

10 M GuSCN: Dissolve 120 g of GuSCN ( mw 118.16) in 100 ml of 0.1 M Tris-HCl, pH 6.4

30 mM EDTA: Add 22 ml of a 0.2 M EDTA solution, pH 8.0 

1.8% Triton X-100: 2.6 g of Triton X-100

Wash Buffer L2

10 M GuSCN : Dissolve 120 g of GuSCN in 100 ml of 0.1 M Tris-HCl, pH 6.4

Procedure

Add 100 ul to tubes containing 50 ul of a suspension of the silica in 0.9 ml of buffer L6.

Final concentration: 

Votex, incubate for 10 min at room temperature

Votex and centrifuge for 15 min at 12,000 g.

Remove supernatant

Wash pellet twice with 1 ml of buffer L2, twice with 1 ml of 70 % ethanol and once with 1 ml of acetone.

Spin each time and remove supernatant

Dry the pellet for 10 min at 56 °C in the thermoblock.

Add 50 ul of sterile water, gently vortex and incubate for 10 min at 56 °C.

Spin and collect supernatant which contains DNA and RNA