title: 16S rRNA Sequencing of microorganisms present in geographically-isolated subglacial environments
design: Extracted DNA from Engabreen, Leverett, Fresh Leverett and Russel enrichments was amplified for the 16S rRNA gene using primers 27F (5-AGA GTT TGA TCC TGG CTC AG-3) and UN1492R (5-TAC GGT TAC CTT GTT ACG ACT T-3) (Lane, 1991). 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. DNA extracted from Finsterwalderbreen and Lower Wright was amplified for the 16S rRNA gene using primers 357F (5-CCT ACG GGG AGG CAG CAG-3) and 518R (5-ATT ACC GCG GCT GCT GG-3') (Muyzer et al., 1993). The PCR reaction mix was kept as described above. For this primer set, template DNA was initially denatured at 94°C for 4 minutes, followed by 35 cycles of denaturing (30 seconds at 94°C), annealing (30 seconds at 54°C) and extension (30 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).