Zymo onestep pcr inhibitor removal kit

DNA from whole 47 mm filters was extracted in a dedicated eDNA laboratory space. DNA was extracted from field samples following the manufacturer’s protocol for the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) and processed using the Zymo OneStep PCR Inhibitor Removal Kit (Zymo Research, Irvine, CA, USA). DNA from experimental samples was extracted using the DNeasy PowerWater Kit (Qiagen, Hilden, Germany), which has been shown to effectively remove PCR inhibitors (Eichmiller, Miller & Sorensen, 2016 (link)). DNA from cartridge filters was extracted using the DNeasy PowerWater Sterivex Kit (Qiagen, Hilden, Germany). The extraction protocol for both PowerWater kits followed the manufacturer’s instructions including the optional heat lysis. The final elution volume was 100 µl into LoBind tubes (Eppendorf, Hamburg, Germany) with an extended incubation time to increase DNA yield (File S5).
Each biological replicate (DNA extracted from a single filter) was assayed for delta smelt in multiple technical replicates (qPCR reactions); eight replicates were used for field samples and six replicates were used for experimental samples. qPCR methods followed the methods described in for assay validation except that a larger volume of DNA template (6.1 µL) was added to each reaction. No-template negative controls and gBlocks positive controls were included on each qPCR plate.

Zymo OneStep^™ PCR Inhibitor Removal Kit (Zymo Research; Cat No./ID: D6030) was used following the manufacturer's protocol in order to remove any carryover PCR inhibitors from previous steps. We added 600 μL of Prep-solution to the column and centrifuged at 8000 g for 3 minutes, the flow-through was discarded, then 50 μL of DNA elute from previous steps were added to the column and centrifuged at 16000 g. The flowthrough was then stored at -20°C.