CTB+N Plot2 Bed3
CTB+N Plot2 Bed3
Metagenomic DNA was extracted from 5 g soil (10 times from 0.5 g) from each of the two beds, the three plots and the ten treatments using the Power Soil DNA Isolation Kit (MO BIO Laboratories, CA, USA), according to the manufacturer’s instructions. The V1-V3 region (about 570 bp) of bacterial 16S rRNA gene was chosen for the amplification and subsequent pyrosequencing. DNA samples were amplified using a set of bacterial primers, the forward 8F (5´-AGA GTT TGA TCI TGG CTC A-3´) and reverse 556R (5´-tta ccg cig cti ctg gca-3´) primers. Each ribosomal primer set was flanked by 454-adapter sequence. Unique 10-nt multiplex identifier was incorporated between the 454-adapter and the forward primer for sample identification among mixed amplicon libraries.  PCR products per soil sample were amplified in quadruplicate, quantified using NanoDrop 2000 (Thermo Fisher Scientific Inc., Suwanee, GA, USA), mixed in equal amount and purified using the DNA Clean and Concentrator™-25 Capped Columns (Zymo Research, Irvine, CA, USA). Sequencing was done by Macrogen inc (Seoul, Korea) on a Roche 454 GS-FLX TitaniumTM pyrosequencer (Roche, Mannheim, Germany).
CTBN13
Cinvestav
CTB+N Plot2 Bed3