Mock community preparation
Twenty-two taxa including the most important planktonic primary
producers (i.e., haptophytes, chlorophytes, bacillariophytes) with some
focus on those taxa relevant in the Arctic were selected from culture
collections (NCMA ex -CCMP or RCC). For details, see Supplementary
Table S1. Five (5) ml of each culture were grown in 50 ml of fresh
K-medium (Keller et al., 1987) for two weeks at 14°C and under light.
Cultures were then filtered onto polycarbonate membrane filters
(Millipore) with a pore size of 0.2 µm, which were stored at -20°C until
extraction.
DNA was extracted with the NucleoSpin Plant II kit (Macherey-Nagel)
following the manufacturer’s protocol. A fragment of the 18S rRNA gene
was amplified using the primer-set 82F (5’-GTGAAACTGCGAATGGCTCAT-3’)
(Šlapeta, Moreira, & López-García, 2005) and 1200R
(5’-GGGCATCACAGACCTG-3’) (Wolf, Kilias, & Metfies, 2014). The PCR
mixtures contained 1 µl of DNA extract, 1 x HotMaster Taq Buffer
containing 2.5 mM Mg2+, 0.8 mM dNTP-mix, 0.2 mM of
each Primer and 0.4 U of HotMaster Taq DNA polymerase in a final volume
of 20 µl. Reaction conditions were as follows: an initial denaturation
at 94°C for 3min, 30 cycles of denaturation at 94°C for 45 sec,
annealing at 59°C for 1 min and extension at 72°C for 3 min, and a final
extension at 72°C for 10 min. DNA concentration of each PCR product was
measured using a Q-bit and converted to copy numbers / µl of PCR
product. Equal amounts of 18S rRNA gene copy numbers from each culture
were mixed together to form the mock community.