Dneasy powerwater sterivex kit

Environmental DNA was extracted from the Sterivex filters by using the DNeasy PowerWater Sterivex Kit (Qiagen, Hilden, Germany) according to a standard protocol with a slight modification as given below. Before the DNA extraction, the DNAiso Reagent was ejected from filter units using a 10 mL disposable syringe. After the addition of incubated MBL Solution to the filters, the filters were further incubated at 65°C on a heat block for 10 minutes to lyse cells and to denaturate proteins in the filtrated residues. In a final elution step of DNA, 25 μL of Microbial DNA-Free Water (Qiagen) was used to avoid unexpected contamination from common elution buffers and reagents. The elution was conducted two times, obtaining a total volume of 50 μL of eDNA solution from each filter unit. The eDNA samples were stored at −30°C after verification of the DNA concentration and quality (OD_260/280 >1.80) using Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific, Waltham, USA).

EPS waste pieces were collected at 7 different sites of varying location, vegetation, and pH, and two control soil samples were also collected from each site, one directly underneath the EPS sample (“S” for soil) and another 0.5 m away (“SN” for soil nearby). The pH values for all soil samples were measured with pH paper of soil mixed with equal parts of water (HYDRION acid-base test paper, Micro Essential Laboratory).
To extract environmental DNA from the EPS samples, the collected pieces were first submerged in no carbon media (26.1 mM Na₂HPO₄, 22 mM KH₂PO₄, 8.6 mM NaCl, 18.7 mM NH₄Cl, 0.4 mM MgSO₄, 36 mM FeSO₄, 29.6 mM MnSO₄, 47 mM ZnCl₂, 4.6 mM CaCl₂, 1.3 mM CoSO₄, 1.4 mM CuSO₄, 0.1 mM H₃BO₃, 34.2 mM EDTA, 1.8 mM HCl), vortexed for 5–10 min, and subjected to DNA extraction with the DNeasy PowerWater Sterivex kit (Qiagen) as instructed by the manual. DNA from soil samples was extracted with the DNeasy PowerSoil Pro kit (Qiagen) following manufacturer’s protocol. These DNA samples were submitted for full length 16S rRNA gene sequencing (forward primer: 5’ AGRGTTYGATYMTGGCTCAG; reverse primer: 5’ RGYTACCTTGTTACGACTT) on the PacBio SMRT platform and the average raw HiFi reads obtained per sample was 16055.

Microbial DNA was extracted from the filters using the DNeasy PowerWater Sterivex Kit (Qiagen, Hilden, Germany) following the manufacturer’s guidelines. DNA concentration and purity were assessed using a spectrophotometer (Eon microplate spectrophotometer, BioTek, Shoreline, WA, USA). Genomic DNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA). Libraries were quantified using the Qubit dsDNA HS Assay Kit and sequenced on an Illumina NovaSeq 6000 platform to generate 150 bp paired-end reads.

The environmental water sample (ES_Ple_Mar) was collected in the Estuary of Plentzia in March of 2023. One and a half liter of water collected from the surface and prefiltered through a 200 μm mesh was sequentially filtered through a 3 μm polycarbonate filters (142mm diameter) followed by a 0.22 μm Sterivex™ filter unit (Millipore) using a MasterFlex Easy-Load peristaltic pump. The sterivex filter with the attached biological material was further used to extract metagenomic DNA following the DNeasy PowerWater Sterivex Kit (Qiagen) protocol. The concentration of the extracted DNA (25.8 ng/μL) was determined using a Qubit 4 Fluorometer (Thermo Fisher Scientific).

DNA from human and soil isolates was extracted from 0.5 ml of EMJH-cultures of single colonies with the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol and used as a template for PCR.
Environmental DNA was extracted from preserved Sterivex filters using the DNeasy PowerWater Sterivex Kit (Qiagen) according to a standard protocol with a slight modification as follows. Before DNA extraction, the Sterivex units were placed at room temperature to dissolve the DNAiso reagent, which was subsequently removed from the units using a 10 mL disposable syringe (Becton, Dickinson and Company). After adding warmed MBL solution, the filter units were further incubated at 65°C on a heat block for 10 min to efficiently lyse the cells and proteins in the filtrated residues. In the final DNA elution, an ultra-clean 25 μL of microbial DNA-free water (Qiagen) was used to avoid the unexpected contamination from common reagents, obtaining a total of 50 μL eDNA solution from each filter unit through an elution step twice. The eDNA was stored at −30°C after verification of its concentration and quality (OD_260/280 >1.80) using a Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific).

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.