Extraction of DNA for qPCR and Cloning

DNA for quantitative PCR (qPCR) and cloning PCR was extracted from cells collected from 1 to 2 mL culture by centrifugation at 4°C and 13,400 rpm for 30 min. DNA was extracted using a modified version of the protocol by Griffiths et al. (2000 (link)) and Urich et al. (2008 (link)), as follows: cells were resuspended in pre-warmed (65°C) 1% sodium dodecyl sulfate (SDS) extraction buffer and transferred to Lysing Matrix E tubes (MP Biomedicals) containing an equal volume of phenol/chloroform/isoamylalcohol (25:24:1). Cell lysis was performed in a FastPrep-24 (MP) device with speed setting 4 for 30 s, and the lysate was centrifuged at 13,400 rpm for 10 min. An equal volume of chloroform/isoamylalcohol (24:1) was added to the supernatant of the lysate, followed by centrifugation at 13,400 rpm for 10 min and collection of the aqueous phase. Nucleic acids were precipitated with an equal volume of isopropanol with addition of 40 μL NaCl 5 M and 1 μL glycogen (20 mg mL⁻¹) as carrier, incubated for 1 h at room temperature. Following centrifugation at 13,400 rpm for 1 h, nucleic acid pellets were washed with 1 mL cold 70% ethanol, dried at 30°C using a SpeedVac centrifuge, eluted in nuclease-free water and stored at −20°C until analysis. Nucleic acid quantification for metagenome sequencing was performed with a Qubit™ 2.0 Fluorometer (Invitrogen) using the dsDNA HS Assay Kit.