IMIS - Marine Onderzoeksgroepen

Study Extent: On 30 January 2006, a mummified crabeater seal was carried from its original resting site near Lake Miers (S 78°05′36″, E 163°51′22″), Miers Valley, Victoria Land, Antarctica, to a new site of similar geomorphology 20 m north. Samples for amplicon sequencing were taken from the original site before transportation (austral summer 2005), the new site before transportation (control, austral summer 2006), and the new site after incubation (austral summer 2009).

Method step description:

1. 16S rRNA gene were generated using the primer pair Tx9 (5′-GGATTAGAWACCCBGGTAGTC-3′) and 1391R (5′-GACGGGCRGTGWGTRCA-3′). PCR was performed in triplicate and pooled to reduce stochastic variability between reactions. Each 30 μl reaction contained 5 or 20 ng of extracted community DNA, Pfx polymerase and platinum polymerase (0.5 U each; Invitrogen), 1× Pfx PCR buffer with Pfx enhancer, 0.2 mM dNTPs, 1 mM MgCl2, 0.02 mg ml−1 BSA, 0.8 μM of forward and reverse primer, and PCR-grade water. Thermal cycling conditions were 94 °C for 2 min; 18 or 24 cycles of 94 °C for 15 s, 55 °C for 30 s and 68 °C for 1 min; and 68 °C for 3 min.
2. Amplicons were size-selected and purified using polyacrylamide gel electrophoresis before being prepared for pyrosequencing by Taxon Biosciences (Tiburon, CA, USA).