DNA Extraction from Sludge and Bacteria

DNA was extracted by standard protocols, including several purification steps for eliminating humid acids that interfere with subsequent molecular techniques [30 (link),36 (link),46 (link)]. A volume of 12 mL of sludge or bacterial suspension was centrifuged for 30 min at 4000× g. The supernatant was discarded, and the pellet was used for DNA extraction using DNeasy^® PowerSoil^® Kit (QIAGEN GmbH., Hilden, Germany) according to the protocol provided by the supplier and a previous study [27 (link)]. The purity, as the concentration of the resulting DNA preparation, was determined spectrophotometrically by the Qubit^® system (Thermo Fisher, Carlsbad, CA, USA). The DNA integrity of molecular weights over 2000 pairs of bases (bp) was evaluated using 2.0% agarose gels (Bioline, London, UK) with Invitrogen SYBR^® Safe DNA gel stain (Thermo Fisher) and TAE buffer solution (40 mol L⁻¹ tris-hydroxymethyl-aminomethane, 20 × 10⁻³ mol L⁻¹ acetic acid, and 1 × 10⁻³ mol L⁻¹ EDTA). Electrophoresis was carried out at 80 mV for 30 min in a Gel XL EnduroTM chamber (Labnet, Edison, NJ, USA) and using 2 µL of DNA per well. Additionally, DNA of E. coli ATCC^® was extracted using an E.Z.N.A^® Bacterial DNA kit (Omega Bio-tek, Norcross, GA, USA), following supplier directions.