| 1. |
Take a clean, broken laboratory glass ware (borosilicate) and grind it using a pestle and mortar until it becomes a fine powder (Wear gloves and facemask). Transfer the finely ground powder to a container and sterilize by autoclaving at 121°C for 15 to 20 min or pre-aliquot and sterilize in amounts that correspond to a standardized procedure. |
| 2. |
Weigh 1 g of soil sample and 1 g of sterile glass powder, transfer in an autoclaved mortar and pestle and grind finely for about 5 min. |
| 3. |
Prepare enough DNA extraction buffer [100 mM Tris, 100 mM EDTA, 1.5 M NaCl (pH 8)]. Sterilize it by autoclaving at 121°C for 15 to 20 min or filter sterilization and store at room temperature. Weigh 10mg (1%) of powdered activated charcoal. |
| 4. |
Add 1 mL of DNA extraction buffer and 10mg of powdered activated charcoal to the soil- glass powder mixture and mix it by pipetting several times. Slowly transfer the contents into a 2 mL eppendorf tube. |
| 5. |
Incubate the tube at 65°C for 10 min in a water bath and then centrifuge at 12000g for 5 min at 4°C. Transfer 500 μL of the supernatant to a fresh 2 mL microfuge tube. |
| 6. |
Prepare enough quantity of 3M sodium acetate (pH 5.2) and 30% PEG (MW-8000). Sterilize by autoclaving at 121°C for 15 to 20 min or filter sterilization and store at room temperature. |
| 7. |
Add 100 μL of sodium acetate and 400 μL of PEG to the supernatant. Allow the mixture to precipitate at -20°C for 20 minutes in a deep freezer. Slowly thaw the tubes and then centrifuge at 12,000g, 5 min at 4°C. |
| 8. |
Discard the supernatant and re-suspend the pellet with 500 μL of autoclaved TE buffer (10 mM Tris, 1 mM EDTA pH 8). |
| 9. |
Prepare chloroform: isoamyl alcohol mixture in the ratio 24:1 and store it in a brown bottle at room temperature. |
| 10. |
Add equal volume (500 μL) of chloroform: isoamyl alcohol mixture and centrifuge at 12,000g for 5 min at 4°C. |
| 11. |
Transfer the aqueous phase to a fresh eppendorf and add 500 μL of ice cold isopropanol. |
| 12. |
Allow precipitation for 5 min at 4°C and centrifuge at 12,000g for 10 min at 4°C. Discard the supernatant and wash the precipitate with 70% ethanol. Centrifuge at 12,000g for 2 min at 4°C. |
| 13. |
Discard the supernatant, air dry the pellet and dissolve in 100 μL of TE buffer (pH 8). |
