Preparation of diatomaceous earth

1, Mix 3 g of diatomaceous earth with 30 ml of sterile milliQ water

2, Let suspensionto settle for 2 to 3 hours, discard upper liquid phase discarded.

3, Resuspend the settled particulate matter in 6.0 ml of sterile milliQ water. This yield stock solution of approximately 150 mg/ml (w/v density of 1.130 to 1.150 g per ml).

4, Add 2.0 ml suspension to 50 ml of chaotropic binding solution (7 M guanidine HCl in 50 mM Tris;
20 mM EDTA; pH 7.0, or 6 M guanidine thiocyanate in 50 mM Tris; 20 mM EDTA; pH 7.0)

Preparation of silica particles.

Silica powder
1, GLASSMILK from BIO101
2, Celite from Sigma/Aldrich
3, 325 mesh powdered flint glass fines from Cutter Ceramics, Beltsville, MD Beltsville, MD, USA.

use the same method as diatomaceous earth for preparing silica powders.

To further remove impurity, add equal vol of concentrated nitric acid and boil in a fume hood, cool down, spin down glass powder, wash extensively with TE buffer and resuspend to give a final concentration of 10 mg/ml. The binding solution should be sodium iodide 908 g/l saturated with sodium sulfte 15 g/l in either 20 mM Tris pH 7.5, or 6 M sodium perchlorate, 50 mM Tris, 10 mM EDTA pH 8.0.

MiniPrep

1. Grow E. coli cells in medium with antibiotics.
2. Spin down cells
3. Resuspend the bacterial cell pellet in 100 ul P1 (25 mM Tris, 10 mM EDTA, 100 ug ml RNaseA, pH 8.0).
4. Add 200 ul of P2 (0.15 M NaOH, 1.0% SDS), mix by inverting.
5. Add 150 ul of N3 (3 M sodium acetate pH 4.8), mix by inverting.
6. Spin 2 X 3.5 min at 4 oC.
7. Mix supernatant with 1.0 ml binding solution, transfer to spin column.
8. Spin
9. Wash metrix twice with 0.35 ml of PE (0 mM Tris; 100 mM NaCl; 2.5 mM EDTA; 55% (v/v) Ethanol; pH 7.5).
10. Allow the packed matrix to completely dry by spinning for another 1~5 minutes.
11, Add 50 ul EB or water, spin to elute DNA.