TITLE: Modified DNeasy PowerWater Kit® protocol for DNA extractions from drinking water samples
AUTHORS: Solize Vosloo, Maria Sevillano, Ameet Pinto

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>DNA-extractions from drinking water samples are essential for a range of subsequent microbial community quantitation and characterization methods, i.e., quantitative polymerase chain reaction (qPCR) assays targeting specific genes and the characterization of compositional and functional profiles using high throughout sequencing technologies (e.g. amplicon sequencing and shotgun metagenomic sequencing). Despite advances in the specificity and sensitivity of molecular techniques, efficient recovery of DNA from drinking water samples, particularly those with low cell counts, remains challenging. Drinking water samples, in which microbial concentrations range between 10</span><span style = "vertical-align:super;">3</span><span>and 10</span><span style = "vertical-align:super;">5</span><span> cells.ml</span><span style = "vertical-align:super;">-1</span><span>, generally requires the collection of large volume of sample and subsequent processing by filtration to concentrate microbial cells. Here we document a modified version of the DNeasy PowerWater Kit® protocol that utilizes enzymatic, chemical, and mechanical lysis strategies to enhance recovery of DNA from drinking water samples. The DNA quantities recovered using this protocol are typically at least two to three-fold higher when compared to the routine DNeasy PowerWater Kit® protocol. In our hands, this protocol consistently provides sufficient DNA of high quality from as little as 1.5 liters of filtered drinking water with cell counts in the range of 10</span><span style = "vertical-align:super;">3</span><span>-10</span><span style = "vertical-align:super;">4 </span><span>cells.ml</span><span style = "vertical-align:super;">-1</span><span>, while maintaining the 16S rRNA qPCR counts at least 100-1000 times higher compared to DNA extracts from negative controls (i.e., blank unused filters, filters with autoclaved deionized water filtered, and reagent blanks) processed identically as the drinking water samples.</span></div><style>
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[STEPS]
?. [PROCESSING OF THE STERIVEX-GP PRESSURE FILTER UNIT]
On the surface of a sterile petri dish (Fisher Scientific, Cat. No: FB0875712), cut the PES filter membrane contained in the Sterivex-GP Pressure Filter Unit (EMD Millipore, Cat. No: SVGP01050) into smaller pieces using a sterile scalpel (Fisher Scientific, Cat. No: 08-920B), and subsequently transfer the cuttings into the 2 ml Lysing Matrix E tube (MP Biomedicals, Cat. No: 116914100) using sterile tweezers (Fisher Scientific, Cat. No: 22327379). For details on how to extract the filter from the Sterivex-GP Pressure Filter Unit, please refer to the following video using this link: https://www.dropbox.com/s/m1ccznfsp02gy2n/IMG_5384.MOV?dl=0
Before handling the Sterivex-GP Pressure Filter Unit (EMD Millipore, Cat. No: SVGP01050), aseptically transfer the ceramic spheres, silica spheres, and glass bead contained in the 2 ml Lysing Matrix E tube (MP Biomedicals, Cat. No: 116914100) to a corresponding 1.5 ml microcentrifuge tubes (Eppendorf, Cat. No: 022431021). Exclusion of these components, essential for downstream mechanical treatment (i.e. bead beating), ensures that the processed polyethersulfone (PES) membrane extracted from filter unit is fully immersed in solution during enzymatic and chemical treatment (STEPS 2 and 3, respectively).
?. [ENZYMATIC TREATMENT WITH LYSOZYME]
Add  10X Tris-EDTA (100 mM Tris, 10 mM EDTA, pH 8.0, G-Biosciences, Cat. No: 501035446) supplemented with 6 μl lysozyme solution (50 mg.ml-1, Thermo Fisher Scientific, Cat. No: 90082) to the 2 ml Lysing Matrix E Tube. Vortex and then incubate for  min at  with light mixing at  using the Eppendorf ThermoMixer C (Eppendorf, Cat. No: 5382000015) making sure that the filter membrane pieces are fully immersed in the solution.
294 µl
37 °C
Final concentration of lysozyme in   solution: 1 mg.ml-1
?. [CHEMICAL TREATMENT AND THE ADDITION OF PROTEINASE K]
Add   PW1 - provided in the DNeasy PowerWater kit and   Proteinase K (20 mg.ml-1, Thermo Fisher Scientific, Cat. No: AM2546). Vortex and then incubate for  min at  with light mixing at  using the Eppendorf ThermoMixer C (Eppendorf, Cat. No: 5382000015) making sure that the filter membrane pieces are fully immersed in the solution.
300 µl
30 µl
56 °C
Final concentration of Proteinase K in   solution: 1 mg.ml-1
?. [CHLOROFORM/ISOAMYL ALCOHOL AND MECHANICAL TREATMENT (BEAD BEATING)]
Aseptically transfer the ceramic spheres, silica spheres, and glass bead contained in the 1.5 ml microcentrifuge tubes (Eppendorf, Cat. No: 022431021) to corresponding 2 ml Lysing Matrix E tube (MP Biomedicals, Cat. No: 116914100).
?. [CHLOROFORM/ISOAMYL ALCOHOL AND MECHANICAL TREATMENT (BEAD BEATING)]
Add  chloroform/isoamyl alcohol 24:1 (Acros Organics, Cat. No: 327155000) and bead beat at setting 6 for  sec using the FastPrep -24™ Classic Instrument (MP Biomedicals, Cat. No: 116004500).
630 µl
Chloroform/isoamyl alcohol treatment should be performed in a laminar fume hood certified for the use of volatile organics. Please refer to the SDS of chloroform/isoamyl alcohol before using it:
?. [CHLOROFORM/ISOAMYL ALCOHOL AND MECHANICAL TREATMENT (BEAD BEATING)]
Centrifuge for  min at  at  and transfer the upper aqueous phase to a clean 1.5 ml microcentrifuge tubes (Eppendorf, Cat. No: 022431021).
4 °C
Expected supernatant recovery range between   and
?. [DNA EXTRACTION USING THE QIACUBE SYSTEM OR QIAGEN HANDBOOK]
Use  of the recovered aqueous phase as sample for the QIACube System (QIAGEN, Cat. No: 9001882). If the sample is less than  add solution PW1 up to the final volume. Follow the instructions as indicated in the DNeasy PowerWater Kit QiaCube Protocol Sheet. If extractions are preformed manually, please continue with steps 11 to 23 as described in the DNeasy PowerWater Kit Handbook.
600 µl
600 µl
?. [DNA EXTRACTION USING THE QIACUBE SYSTEM OR QIAGEN HANDBOOK]
Store the DNA at   until further processing.
-80 °C