Timepoint 5 - Dynamical changes of soil microbiota during a one-year period timeframe and crop rotation under No Till farming
DNA was isolated from 10 g of soil using the PowerMaxSoil DNA Isolation Kit (MO BIO Laboratories, Inc, USA). The hyper variable V4 region of the 16S rRNA gene was amplified using the following universal primers containing the Roche 454 sequencing A and B adaptors (underlined) and a 10 nucleotide “multiple identifier” (MID) to sort samples (bold): 520F (5’-CGTATCGCCTCCCTCGCGCCATCAGACGAGTGCGTAYTGGGYDTAAAGNG -3’, were ACGAGTGCGT is an example, different MIDS for each sample were used) and 802R (5’-CTATGCGCCTTGCCAGCCCGCTCAGTACCRGGGTHTCTAATCC, 5’-CTATGCGCCTTGCCAGCCCGCTCAGTACCAGAGTATCTAATTC, 5’-CTATGCGCCTTGCCAGCCCGCTCAGCTACDSRGGTMTCTAATC 5’-CTATGCGCCTTGCCAGCCCGCTCAGTACNVGGGTATCTAATCC) (modified from RDP’s Pyrosequencing Pipeline: http://pyro.cme.msu.edu/pyro/help). The samples were multiplexed in the following way: Region 1: t5A (ACGAGTGCGT); t5N (TCTCTATGCG).