IESTPLM01__MD3304D08C__18S-F
DNA was extracted using two-step alkaline DNA extraction protocol that were conducted with 0.1 g (wet weight) of marine sediments, alkaline solution (Kouduka et al., submitted). DNA from living organisms was removed by the first extraction under mildly heated conditions at 65 degrees Celsius, which was followed by the second extraction at 95 degrees Celsius.
The 18S rRNA gene was amplified using the primers EukV4F (CCATCTCATCCCTGCGTGTCTCCGACTCAG-MID-CCAGCASCYGCGGTAATTCC) and EukV4R (CCTATCCCCTGTGTGCCT-ACTTTCGTTCTTGATYRA) (Oris et al., 2013). The MID is a multiplex identifier sequence which allows pooling of amplicons from different individuals (Hamady et al., 2008). A reaction mixtures was prepared in which the concentration of each oligonucleotide primer was 0.1 uM and that of the DNA template was ca. 0.1 ng/uL. Thermal cycling with the primers EukV4F and EukV4R was performed with 35?40 cycles of denaturation at 96 degrees Celsius for 20 s, annealing at 56 degrees Celsius for 45 s and extension at 72 degrees Celsius for 120 s. Thermal cycling for the V4 region was performed with 40 cycles of denaturation at 96 degrees Celsius for 20 s, annealing at 50 degrees Celsius for 45 s and extension at 72 degrees Celsius for 120 s.