title: Otumba soil sample plus Anthracene day 14 replicate 3
design: The soil sample was washed first with 0.15 M sodium pyrophosphate and 0.15 M phosphate buffer pH 8 to remove the organic material before the DNA was extracted (Ceja-Navarro et al., 2010). Three different methods were used to extract the DNA from soil. A first technique used a chemical and thermal shock for cell lysis (Ceja-Navarro et al., 2010). In a second technique cells were enzymatically lysed (Sambrook and Rusell, 2001) and in a third technique a detergent solution and mechanic disruption were used for cell lysis (Hoffman and Winston, 1987). Each technique was used to extract four times 0.5 g soil (a total 2 g soil) and pooled. As such, a total of 6 g soil was extracted for DNA.    Primers 8–F (5´–AGA GTT TGA TCI TGG CTC A– 3´) and 556–R (5´ –TGC CAG IAG CIG CGG TAA– 3´), 10 pb multiplexed and containing the Roche 454 pyrosequencing adaptors Lib–L, were used to amplify the region V1–V3 of the 16S rRNA gene from the metagenomic DNA. Details of the PCR mixture and the thermal cycling scheme can be found in Navarro–Noya et al. (2013). All samples were amplified five times, pooled in equal amounts, and purified using the GFXTM  PCR DNA an Gel Band Purification Kit (GE Healthcare, UK) following the manufacturer instructions. Quantification of the PCR products was done using the PicoGreen dsDNA assay (Invitrogen, Carlsbad, USA) in a NanoDropTM 3300 fluoroespectrometer (Thermo Scientific NanoDrop).