Metabarcoding of environmental samples from littoral water of lake Dagowsee
Frozen Sterivex cartridges were broken open and half of the fragmented filter was transfered into each of two 2-ml tubes. Samples were then incubated for 1 hr at 65 C, centrifuged, the supernatant washed with an equal volume of chloroform-IAA, precipitated with 2 volumes of PEG/NaCl (30% PEG 6000 in 1.6M NaCl), cleaned with ice-cold 70% ethanol, and eluted in 50 l nuclease free water. Subsamples were pooled to give 100 l nucleic extract per sample. RNA was removed by the addition of 0.5 l (5g) RNase A (10mg/ml DNase and protease free) to 80 l of the pooled sample, incubated at 37C for 30 minutes, and cleaned up using the PowerClean Pro DNA Clean-Up kit (MoBio). PCRs were conducted in 25-l reactions using 0.5 l Herculase II Fusion enzyme (Agilent Technologies), 5 l of 5x PCR buffer, 0.62 l each primer (10 uM), 0.25 l dNTPs (250 mM each), 0.3 l BSA (20mg/ml BSA) on a SensoQuest labcycler with 2 min denaturation at 95 C, 13-30 cycles of 94 C for 30 sec, 55 C for 30 sec and 70 C for 4 min, and a final elongation at 70 C for 10 min. Multiple PCR reactions (up to 50) were required for each environmental sample to ensure sufficient product for library preparation (1g purified PCR product).