Illumina Sequencing: Microbial diversity of anodic biofilm in Microbial fuel cell inoculated with activated sludge
The DNA was extracted and was run on 1% agarose, for quality and quantity check. Also quantification was carried out by Nanodrop. 25ng of DNA was used to amplify 16S rRNA hyper variable region V3-V4. The reaction includes KAPA HiFi HotStart Ready Mix and 100 nm final concentration of modified 341F and 785R primers with partial Illumina sequencing adapters overhangs. The PCR involved an initial denaturation of 95C for 5 min followed by 30 cycles of 95C for 30s, 55C for 45s and 72C for 30s and a final extension at 72C for 7 min. The amplicons were purified using Ampure beads to remove unused primers. Additional 8 cycles of PCR was performed using Illumina barcoded adapters to prepare the sequencing libraries.