454-sequencing analysis of activated sludge samples from textile and municipal wastewater treatment plants
PCR amplification was performed in duplicate on each pooled DNA sample in a reaction volume of 20 ľl, containing 1.0 ľl 10x diluted genomic DNA, 1.5 ľl dNTP mixture (2mM stock; Invitrogen, Carlsbad, CA, USA), 0.5 ľl of each primer (20 ľM stock), 2.0 ľl 10x Titanium Taq PCR buffer, 0.4 ľl Titanium Taq DNA polymerase and 14.1 ľl nuclease-free water. PCR conditions were as follows: initial denaturation of 2 min at 94°C, followed by 30 cycles of denaturation at 94°C for 45 s, annealing at 61°C (bacteria) or 63°C (archaea) for 45 s and extension at 72°C for 60 s, and a final extension step for 10 min at 72°C. Following agarose gel electrophoresis, amplicons of the expected size range were excised and extracted from the gel using the QIAquick gel extraction kit, according to the manufacturers instructions. Purified dsDNA amplicons were quantified using a Qubit 2.0 fluorometer and the high-sensitivity DNA reagent kit, and diluted to equimolar concentrations.