Soil Microbiome DNA Extraction

In a 2-ml screw cap tube containing 500 mg of 0.1-mm silica spheres (MP Biomedical, Solon, OH, USA), 200 mg of cecal content, and 700 μl of lysis buffer [Tris-HCl 500 mM pH 8, EDTA 100 mM pH 8, NaCl 100 mM, SDS 1% (w/v)] were mixed together. A 900-μl volume of lysis buffer was used as a negative control. A mechanical lysis step was performed using a FastPrep-24™ 5G Instrument (MP Biomedical) for three runs of 60 s each, at 6 m/s. Samples were kept on ice during 5 min between each run. A second step involving thermal lysis was carried out on the samples that were heated for 20 min at 95°C and kept for 5 min on ice at the end of the procedure. The supernatant was collected after a centrifugation at 18,000 × g for 15 min and a standard phenol/chloroform purification protocol was used to complete the DNA extraction (11 (link)). The DNA concentration of each sample was measured using a QFX Fluorometer (Froggabio, Toronto, ON), and the purity of those samples was assessed using a Nanodrop 1000 (Fisher, Ottawa, ON) device. DNA samples were stored at −20°C until analysis.