166700F156516_S44
cover crop soil metagenome
The microbial communities was investigated by the bacterial and fungal automated ribosomal intergenic spacer analysis (B-ARISA and F-ARISA respectively), using the bacterial primers ITSF/ITSReub (Cardinale et al., 2004) and the fungal primers 2234C/3126T (Ranjard et al., 2001). The PCR fragments were labelled as described in Schuelke, 2000. For each reaction three primers was used: a sequence-specific forward primer with M13 tail at its 5 end; a sequence-specific reverse primer not modified, and the universal fluorescent-labeled M13 primer. PCR mixtures (30 L) contained buffer Phire Hot Start 1X (Thermo Scientific), dNTP 0,2 mM, sequence-specific forward primer with M13 tail 0,8 M, sequence-specific reverse primer 0,3 M, the universal fluorescent-labeled M13primer 0,3 M, BSA 0,1% (Biolabs), Phire Hot Start II DNA Polymerase (Thermo Scientific) 0.4 l, 20 ng of genomic DNA extracted by soil samples . The ITS bacterial region was amplified as follows: 98C for 30 s, followed by 35 cycles consisting of 98C for 15 s, 50C for 15 s, 72C for 30 s.; 72C for 1 min. ITS eukaryotic region was amplified in the same conditions except in  the annealing step, where thetemperature was 58C. PCR products (5 l) were loaded on a 2% agarose gel and separated by electrophoresis. Gels were stained with 1% ethidium bromide.
166700F156516_S44
SUB7081733
166700F156516_S44