Metabarcoding of environmental samples from the littoral water of lake Stechlin
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 (5 g) RNase A (10mg/ml DNase and protease free) to 80  l of the pooled sample, incubated at 37 C 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 (1 g purified PCR product).