title: 16S rRNA sequencing of microrganisms present in Fe(III)-reducing ISA degrading enrichment culture.
design: Extracted DNA from Fe-reducing-ISA enrichment cultures was amplified for the 16S rRNA gene using primers 27F (5-AGAGTTTGATCCTGGCTCAG-3) (Lane 1991) and 907R (5-CCGTCAATTCMTTTRAGTTT-3) (Muyzer 1995). Each 50?l PCR reaction mixture contained 1?l DNA template, primers (0.4?M each), deoxynucleoside triphosphates (dNTPs; 200?M), 1.5mM MgCl2, 5?l 10X PCR buffer, 5?l bovine serum albumin (BSA), and 0.5?l Taq polymerase. Template DNA was initially denatured at 94°C for 4 minutes, followed by 30 cycles of denaturing (30 seconds at 94°C), annealing (30 seconds at 55°C), and extension (60 seconds at 72°C), and a final extension at 72°C for 5 minutes. Amplified DNA was sequenced for Bacterial 16S ribosomal RNA using the Roche 454 Pyrosequencing platform (Research and Testing Laboratories, Austin, TX, USA).