Ampure xp pcr purification

DNA samples were amplified with 454-fusion primers Adapter B/ITS3 and ITS4/6-bp tag/ Adapter A (Rajala et al., 2015 ), targeting the fungal rDNA ITS2 region. Polymerase chain reaction (PCR) mixes contained 9.9 μl sterile water, 5 μl 5 × Phusion GC buffer (Thermo Fisher Scientific), 0.5 μl dNTP mix (10 mm), 1.2 μl of each primer (8 μm), 0.8 μl DMSO, 0.2 μl Phusion DNA Polymerase (Thermo Fisher Scientific) and 1 μl DNA template, in a final volume of 20 μl. The optimal number of PCR cycles was tested for each sample to reach clearly visible but relatively weak bands in agarose gel (5 μl of PCR product). When needed, dead wood samples were further purified with polyethylene glycol precipitation (Vainio et al., 1998 ), and soil samples were diluted 1:4 or 1:3. Final amplification was then performed using the following cycling conditions: 30 s at 98 °C; 20–28 cycles of 10 s at 98 °C, 30 s at 55 °C and 30 s at 72 °C; and a final extension at 72 °C for 5 min.
PCR products were purified with AMPure (Agencourt AMPure XP PCR purification, Beckman Coulter, Brea, CA, USA), and concentrations were measured with a Qubit 2.0 fluorometer using a Quant-iT dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA). Bioanalyser (DNA1000 chip, Agilent Technologies, USA) was further used to ensure the quality of purified PCR products.