Ecological Archives E096-082-A3
Cyrus A. Mallon, Franck Poly, Xavier Le Roux, Irene Marring, Jan Dirk Van Elsas, and Joana Falcão Salles. 2015. Resource pulses can alleviate the biodiversity–invasion relationship in soil microbial communities. Ecology 96:915–926. http://dx.doi.org/10.1890/14-1001.1
Appendix C. Quantification of initial species richness with 454 pyrosequencing in the removal experiment.
Using the same extracted DNA that was used for 16S quantitative PCR as a template, libraries of individual samples from Day 0 were amplified with forward primer (5'-TGYCAGCMGCCGCGGTA-3') and reverse primer (5'TCACGRCACGAGCTGACG-3') with a unique 10 base pair molecular identification tag for each sample. One 25 µL PCR reaction contained 2.5 µL of 10X PCR Buffer, 1.8 µL of 25 mM MgCl2, 0.5 µL of dNTPs, 0.25 µL of 20 mg/mL Bovine Serium Albumin, 0.5 mM of each primer with an initial concentration of 10 uM, and 0.25 µL of high fidelity taq polymerase (Roche Diagnostics GmbH, Mannheim, Germany). The reaction was adjusted with water to reach 25 µL. The PCR reaction began with an initial denaturing step for 5 minutes at 95°C, followed by 30 cycles of denaturation for 40 seconds at 95°C, annealing for 45 seconds at 60°C, and elongation for 30 seconds at 72°C. The reaction was completed with a final elongation step of 10 minutes at 70°C.
PCR products were then visualized via staining with ethidium bromide on a 2.5% agarose gel that had run for 5 hours at 70 volts. 16S bands were excised from the gel and DNA was extracted and purified using the QUIquick Gel Extraction Kit according to the manufacturer's instructions (Quiagen GmbH, Hilden, Germany). DNA quantity of purified PCR products were then quantified using the PicoGreen dsDNA flourescece assay (Invitrogen, Paisley, UK), equimollarly pooled, and sent for sequencing via the Roche GS-FLX pyrosequencer with titanium chemistry at Beckman Coulter Genomics (Danvers, MA, USA).