Microbial Diversity Assessment through DNA Extraction, PCR, and DGGE

Extraction, PCR and denaturing gradient gel electrophoresis were undertaken as described in [16 (link)]. DNA was extracted from all samples using QIAGEN DNeasy blood and tissue kits, and bacterial 16S rRNA gene diversity was amplified using primers 357F and 518R. PCR protocol was as in [17 (link)]. Ciliate 18S rRNA gene diversity was amplified using primers CilF and CilDGGE-r. PCR protocol was as in [17 (link)]. For each of the above primer pairs, 30 µl PCR mixtures containing 1.5 mM MgCl₂, 0.2 mM dNTP (Promega), 0.5 mM of each primer, 2.5 Ul of Taq DNA polymerase (QBiogene), incubation buffer and 20 ng of template DNA [17 (link)]. DGGE was performed as in [17 (link)] using the D-Code universal mutation detection system (Bio-Rad). PCR products were resolved on 10% (w/v) polyacrylamide gels for bacterial 16S rRNA gene diversity and 8% (w/v) for ciliate diversity. Bands of interest (those which explained the greatest differences/similarities between samples) were excised from DGGE gels, re-amplified with the same original primers, labelled using a big dye (Applied Biosystems) transformation sequence kit and sent to Genevision (Newcastle University, UK) for sequencing.