Direct seeding with crop rotation plot 3 Control day 1
Three different techniques were used to extract the DNA from 2 g soil. The first method used was based on the technique described by Ceja-Navarro et al. (2010) and consisted in a chemical and thermal shock for cell lysis. Cells were enzymatically lysed in the second method (Hofman and Winston, 1987), while a detergent solution and mechanic disruption for cell lysis was used in the third method (Sambrook, 2001).  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 barcoded 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. The PCR mixture and the thermal cycling were done as described by Navarro-Noya et al. (2013). All samples were amplified in triplicate, and purified using the GFXTM PCR DNA an Gel Band Purification Kit (GE Healthcare, UK). Quantification of the PCR products was done using PicoGreen dsDNA assay (Invitrogen, Carlsbad, USA) and a NanoDropTM 3300 fluoroespectrometer (Thermo Scientific NanoDrop) and pooled in equimolar amounts. Pools of 50 purified amplicon-libraries were sequenced.
Cinvestav
DSMS3CD01