Cultured community from the rhizosphere of a mine tailing established Acacia farnesiana on patch 2
Bacterial colonies were tooth-picked into a 2 ml centrifuge tube for each community containing sterilized water, then proteinase K and lysozyme (both from Sigma-Aldrich, St. Louis Missouri, United States) were added for an initial lysis (37C, 30 min). Then the tubes  were centrifuged and the pellets were used as the source for DNA extraction with the MoBio PowerSoil extraction kit. PCR reactions were carried out to amplify the V3-V4 region (341F and 805R primers; 464 bp amplicon; Klindworth 2013) following the Illumina  MiSeq protocol with 5 overhangs. We performed triplicated PCR reactions for each sample, using the high fidelity Pfx platinum polymerase (Invitrogen, Thermo Fisher Scientific Corporation, Carlsbad, California, USA). Finally, amplicons from each replicate were pooled and purified with the Wizard SV Gel and PCR Cleanup System kit (Promega Corporation, Madison, Wisconsin, USA).