16S rDNA of anaerobic digested sludge
Microbial DNA was extracted from sludge samples using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturers protocols. The V4-V5 region of the bacteria 16S ribosomal RNA gene were amplified by PCR (95 C for 2 min, followed by 25 cycles at 95 C for 30 s, 55 C for 30 s, and 72 C for 30 s and a final extension at 72 C for 5 min) using primers 515F 5-barcode- GTGCCAGCMGCCGCGG)-3 and 907R 5-CCGTCAATTCMTTTRAGTTT-3, where barcode is an eight-base sequence unique to each sample. PCR reactions were performed in triplicate 20 L mixture containing 4 L of 5  FastPfu Buffer, 2 L of 2.5 mM dNTPs, 0.8 L of each primer (5 M), 0.4 L of FastPfu Polymerase, and 10 ng of template DNA. Amplicons were extracted from 10% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturers instructions.
SUB14714926
R4-B