DNA Extraction from Environmental Samples

DNA was extracted from half of each filter using a modified Miller protocol^{32 (link)}. Half of each filter was placed into a Lysing Matrix E tube (MP Biomedicals) along with 300 µl of Miller Phosphate buffer, 300 µl of Miller SDS lysis buffer, and 600 µl of Phenol:Chloroform:Isoamly alcohol (25:24:1). A process blank was also setup and subjected to all following steps of extraction without a filter. The tubes were homogenized in a FastPrep-24 bead-beater (MP Biomedicals) for 45 seconds at a speed of 5.5 m/s. To remove cell debris and filter material, the tubes were centrifuged at 10,000 × g for 5 minutes. 600 µl of aqueous supernatant was transferred to a new 2 ml tube along extracted with one volume of chloroform. Tubes were centrifuged at 10,000 × g for 5 minutes, then the aqueous phase was kept. Purification and concentration of recovered DNA was performed by adding two volumes of MoBio solution C4 to the aqueous phase. This was passed over a MoBio spin filter. The spin filters were then washed with 400 µl of MoBio C5 solution. Residual C5 was removed by centrifuging the empty tubes at 10000 × g for two minutes. DNA was eluted by two 30 µl additions of MoBio C6 solution. Final eluted environmental DNA was stored in −80 °C freezer. DNA concentrations were determined using NanoDrop spectrophotometer.