ITS1 amplicons from soil and B. pubescens roots
Two 3 mm tungsten carbide beads were added to each 2 ml Eppendorf tube containing roots in 600 l CTAB, or 600 l soil/CTAB sludge (both roots and soil were treated using the same protocol) and these were then grinded using TissueLyser II (Qiagen, Hilden, Germany) for one minute at 25 Hz, two times for each tube. After grinding, the samples were incubated for 30 min at 65 C. Once cooled to room temperature, 600 l chloroform was added to each tube and the solution was mixed with a vortex. The tubes were then centrifuged at 13,200 rpm for the layers to separate. 400 l of the upper layer was transferred over to tubes containing 400 l isopropanol (taken straight out of a -20 C freezer). These tubes were inverted several times, and the mixture was allowed to sit for 1 hour or more for the DNA to precipitate. These tubes were then centrifuged at 13,000 g for 10 min to make a compact DNA pellet. The supernatant was discarded, and the DNA was eluted in 200 l E.Z.N.A elution buffer (Omega Bio-tek, Norcross, USA). Cleanup of the DNA pellet was done with E.Z.N.A. Soil DNA Kit, following the manufacturer's protocol (Omega Bio-tek, Norcross, USA). Each session of extraction consisted of 23 samples plus one extraction negative. Technical replicates were introduced in the PCR step. Due to limited space on the 96-well plates the extraction negatives were amplified separately and checked for DNA using 1% agarose gel. The ITS1 region of rDNA was targeted using ITS-1F (forward) and ITS2 (reverse) primers (White et al., 1990; Gardes and Bruns, 1993). All primers were tagged with molecular identifiers for retrieval in downstream bioinformatics. Each PCR reaction consisted of 1l DNA template and 24 l master mix: 15.7 l milli-Q H2O, 2.5 l GoldBuffer, 2.5 l GoldMgCl, 0.2 l 25 nmol dNTP, 1 l 20 ng/ml BSA, 0.125 l AmpliTaq Gold, 1l 10 mol forward primer and 1l 10 mol reverse primer. Ten samples amplified poorly after two tries and were then amplified using the same mastermix but with 3 l DNA template instead of 1 l. PCR reactions for ITS1 were run with initial denaturation at 95 C for 5 min, followed by 32 cycles of denaturation at 95 C for 30 sec, primer annealing at 55 C for 30 sec and elongation at 72 C for 1 min. An elongation step was included after the cycles at 72 C for 7 min, before cooling down to 4 C. Two PCR negatives, one mock community and three technical replicates were included on each plate of 96 samples. Each PCR product was controlled for positive amplification with gel electrophoresis using a 1% agarose gel, before individual clean-up and purification of the amplicons with ZR-96 DNA Clean & Concentrator-5 kit (Zymo Research, California, USA).