16S rRNA sequencing of rhizosphere soil: lettuce cultivated in soil with hairy vetch cover crop replicate 1 at mature stage of plant
The first amplification was done using the primers 515F and 806R in PCR conditions: 95 for 10 min; 95 for 30 sec / 57 for 30 sec / 72 for 1 min (20 cycles); 72 for 7 min. PCR reaction (total 20 l): 10 L AmpliTaq Gold 360 Master Mix (AB Applied Biosystems, USA), 0.4 L of each primer, 1 L of template DNA, and 8.2 L of nuclease-free water. The product was purified using AMPure XP reagent (Nipon Genetics Co. Ltd., Japan). The second amplification was done to the purified first PCR product in 20 l of total PCR reaction using the same primers attached with barcodes and adaptor in PCR condition: 95 for 10 min; 95 for 30 sec / 57 for 30 sec / 72 for 1 min (5 cycles); 72 for 7 min. Products are pooled equimolar to a final concentration of 50 pM and sequenced using the Ion PGM Hi-Q Chef kit.