Community DNA Extraction from Bacterial Colonies

1. Bacterial Community DNA Extraction

1. To begin the procedure, weigh out 100 g of sieved soil. Add this to a polypropylene vessel, and add 100 mL of extraction buffer comprised of Tris buffer amended with EDTA to promote the release of bacteria from the soil matrix, then shake by hand.
2. Next, weigh 100 g of glass beads, and add these to the mixing vessel. Agitate the sample for 5 min using a bead beating device or mechanical wrist-action shaker for 15 min. Add 10 mL 20% sodium dodecyl sulfate, or SDS, to the mixture, then agitate for an additional minute. Incubate at a high temperature of 60 - 65 °C for 60 min.
3. Equally distribute the sample among separate 50-mL tubes, and centrifuge for 10 min at 6,000 x g. Transfer the supernatant from the tubes to a single sterile container. Next, repeat the extraction on the soil pellet as previously described, using a fresh volume of extraction buffer.
4. Next, add the total volume of processed supernatant, approximately 200 mL, to a clean 50-mL tube filled to half volume with a solution of 30% polyethylene glycol and 1.6 M sodium chloride. Invert the bottles several times by hand to mix, and incubate at room temperature for 2 h. Centrifuge samples at 10,000 x g for 20 min to pellet the DNA.
5. Remove the supernatant carefully from the centrifuge tube, leaving behind the partially purified nucleic acid pellet. Add 20 mL of TE Buffer and 1.5 mL of a 7.5 M potassium acetate solution to resuspend the pellet, then vortex. Place the suspension on ice for 5 min. Centrifuge at 16,000 x g for 30 min at 4 °C to precipitate proteins and polysaccharides.
6. Next, add an RNAse and proteinase K to the sample, mix gently by hand, and let sit for moment. Add an equivalent volume of phenol:chloroform:isoamyl alcohol (ratio mixture of 25:24:1) to the suspension to be extracted, and mix gently by hand. Centrifuge the preparation for 10 min at 13,000 x g. Carefully remove the vessel from the centrifuge, and note the two layers.
7. The bottom, heavier layer is comprised of the phenol:chloroform:isoamyl alcohol and extracted debris, and the top layer is the aqueous and contains the DNA. Place the aqueous phase into a sterile vessel, add an equivalent volume of isopropanol, and invert gently to initiate DNA precipitation. Incubate the suspension at room temperature for 2 h. Pellet the purified DNA by centrifugation at 16,000 x g for 30 min. Carefully remove the supernatant as the pelleted DNA may or may not be visible at the bottom of the vessel, and then resuspend in 1 mL of TE Buffer.
8. Using a spectrophotometer or DNA/RNA quantification fluorimeter, measure the level of DNA extracted from the sample. The amount of DNA is estimated from the 260 nm reading. An absorbance reading of 1.0 is equivalent to 50 µg of DNA per mL of solution. If the concentration is too high for accurate readings, dilute the suspension 1 to 10, or 1 to 100 using molecular grade water.
9. The purity of DNA is estimated from the ratio of the reading at 260 nm to that at 280 nm. A value > 1.7 indicates relatively pure DNA. The maximum theoretical value is 2.0.