Antarctic cryosphere fungal diversity (2015)

Study Extent

Sampling

Method steps

1. The samples were melted and a total of 12–15 L of the resulting water Filtered through 47 mm diameter (Millipore) membranes (three membranes per sampling site, each using 4–5 L) until each membrane became saturated. Membranes were then stored at -20°C until DNA extraction.
2. The three membranes resulting from filtering the melted ice from each sampling site were processed together in order to increase DNA yield. Total DNA was extracted using 0.5 mL extraction buffer [sodium dodecyl sulfate (SDS) 10%], left at 55°C for 18 h, followed by 165 μL NaCl (5 M) and 165 μL cetyltrimethylammonium bromide (CTAB, 10%), then 600 μL chloroform was added and the mixture centrifuged (Eppendorf/Germany) at 13,000 rpm for 10 min. The supernatant was cleaned using the QIAGEN DNeasy PowerClean cleanup Kit. The ITS2 region was used as a DNA barcode, using the universal primers ITS3 and ITS4 and were sequenced at Macrogen Inc. (South Korea) on an Illumina MiSeq sequencer, using the MiSeq Reagent Kit v3 (600-cycle) following the manufacturer’s protocol.