Use of PCR-DGGE Based Molecular Methods to Analyse Microbial Community Diversity and Stability during the Thermophilic Stages of an ATAD Wastewater Sludge Treatment Process as an Aid to Performance Monitoring

Table 3

Optimized conditions for DGGE analysis.

Amplicon	Primers set	Optimal parameters (DGGE analysis)
		Amount of DNA template in PCR reaction	Voltage, V	Time, hrs	Denaturant range, (%)	Amount of amplicon (per lane), μg	Gel staining methods
V3–V5; 16S rRNA	338F-GC∗/518R	300 ng	75	16	35–75	4.5	EtBr
V6–V8, 16S rRNA	948F-GC∗/L1401	300 ng	75	18	40–65	1.5; 3	Silver stain; EtBr
rpoB	1698F-GC∗/2043R	600 ng	75	16	30–60	0.3	EtBr, silver stain

∗GC clamp added to the 5′ end of the primers 338F, 948F, and 1698F, 5′CGCCCGCCGCGCGCGGCG GGCGGGGCGGGGGCACGGGGGG-3′.