Ecological Archives E096-082-A2
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 B. Quantification of bacterial abundance in the removal experiment via quantitative PCR.
DNA was extracted using 0.5 g of soil with the powersoil DNA extraction kit (MOBIO, Carlsbad, California, USA) according to the manufacturer's instructions, except that 0.25 g of 1 mm glass beads were added in the first step in order to aid in cell lysis. DNA concentration was quantified using PicoGreen dsDNA assay (Invitrogen, Paisley, UK). The V5-V6 region of the 16S rRNA gene was amplified using forward primer (3'-GGTAGTCYAYGCMSTAAACG-5') and reverse primer (3'-GACARCCATGCASCACCTG-5') (Bach et al. 2002). For each reaction, 12.5 µL of Power SYBRgreen master mix (Applied Biosystems, Carlsbad, California, USA) was added with 0.75 µl of each primer, 10 µL of ultrapure water, and 1 µL of template DNA. The cycling program began with a 10 minute denaturation phase at 95 °C, followed by 40 cycles of denaturation at 95 °C for 27 seconds, annealing at 62 °C for 1 min, and extension at 72 °C for 30 seconds. Quantification was carried out in the ABI Prism 7300 Cycler (Applied Biosystems, Foster City, California, USA). The standard curve came from plasmid DNA, where the 16S rRNA gene from Serratia plymuthica was cloned into the pGEM-T plasmid was used as a standard template with concentrations ranging from 106 to 101 copies / µL.
Bach, H., J. Tomanova, M. Schloter, and J. Munch. 2002. Enumeration of total bacteria and bacteria with genes for proteolytic activity in pure cultures and in environmental samples by quantitative PCR mediated amplification. J. Microbiol. Methods 49:235-245.
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