IMIS - Marine Onderzoeksgroepen

Colonized quartz stones were retrieved by hand (using isopropyl alcohol surface sterilized latex gloves) and loose soil particles gently removed with a sterile (autoclaved) paintbrush. Samples were then stored in sterile Whirlpak bags (Nasco) at -20°C in the field and in transit, and subsequently stored frozen at -80°C in the laboratory until processed.
Study Extent: Hypolithic communities were recovered from quartz substrate in desert pavement on the Tibetan Plateau (China), the Taklimakan Desert (China), Devon Island (Canada) and the McMurdo Dry Valleys (Antarctica).
Method step description:

1. Amplification of 16S rRNA genes was achieved using primer pair 341F and 907R with PCR conditions involving an initial denaturation time of 5min; 30 cycles at 95°C for 1 min, 55°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 10 min. Positive and negative controls were run for every PCR. For each amplicon library purification was carried out with Agencourt AMPure XP Bead (Beckman Coulter, CA, USA) according to manufacturer’s instructions. The libraries were quantified with Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen Life Technologies, NY, USA) using FLUOstar OPTIMA F fluorometer (BMG Labtech GmbH, Offenburg, Germany) and library quality was assessed with the FlashGel System (Lonza Group Ltd., Basel, Switzerland).
2. Emulsion-PCR was carried out with GS Junior Titanium emPCR Kit (Lib-L, 454 Life Sciences Corp., CT, USA) according to the emPCR Amplification Method Manual – Lib-L, Single-Prep. The sequencing reaction was carried out with the GS Junior Titanium Sequencing Kit and GS Junior Titanium PicoTiterPlate Kit (454 Life Sciences Corp.) according to the manufacturer’s instructions. The sequencing run was conducted in 200 cycles.