title: Metabarcoding of extraction blank
design: Extraction blank using 300 mL of dH20 following in place of recombined bulk community sample was performed using a modified protocol for the E.Z.N.A Blood Maxi Kit (OMEGA Bio-Tek, Georgia, USA). A clean 63-µm Nitex filter via a polycarbonate filter holder and gentle hand-operated vacuum pump followed by backflushing with 0.5 mL of CSPL buffer (Omega Bio-Tek, Georgia, USA). Zooplankton were finely ground by pestle and 4.5 mL of additional CSPL buffer was added with 250µL of Proteinase K. Samples were vortexed and incubated overnight in a 55°C water bath. Subsequent steps followed the manufacturer's protocol. Additional modifications included warming the elution buffer to 70°C to enhance DNA recovery, and minimizing the elution volume to 1.1 mL for the first elution. The first elution was concentrated using the DNA Clean & Concentrator kit (Zymo Research). The V1-V2 region of the nuclear 18S rRNA gene (18S V1-V2) using primers F04: 5' GCTTGTCTCAAAGATTAAGCC 3' and R22mod: 5' CCTGCTGCCTTCCTTRGA 3' (∼365 bp; (Fonseca et al. 2010; Sinniger et al. 2016) was amplified using polymerase chain reaction (PCR). Each reaction contained 3 µL of template DNA, 12.5 µL of MyFi™ Mix (Meridian Bioscience), 1 ng/µL Bovine Serum Albumin (0.5 µL), and 10 µM forward and reverse primers (0.5 µL each), with molecular grade H20 added for a total reaction volume of 25 µL. PCR conditions were initial denaturation at 95 °C (2 min), followed by 40 cycles of denaturation at 95 °C (45 sec), annealing (55 °C for 45 sec), and extension at 72 °C (1 min), followed by final extension at 72 °C (10 min). PCR amplification was conducted in duplicate and products were pooled for library preparation. Pooled amplicons were purified using AMPure® XP beads (Beckman Coulter®), quantified using a Qubit® 1X dsDNA High Sensitivity Fluorescence kit (Thermofisher Scientific), and normalized to a concentration of 4ng/µL with molecular grade H20. Aerosol barrier pipette tips were used throughout DNA extraction, PCR, and library. Aerosol barrier pipette tips were used throughout DNA extraction, PCR, and library, additionally filters, filter holders and tissue grinders were treated with 10% bleach overnight, 3x dH2O rinsed, and dried prior to use to minimize contamination.