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URL: https://dx.doi.org/DOI:10.17504/protocols.io.66khhcw

Authors: Solize Vosloo, Maria Sevillano, Ameet Pinto

Group: Pinto Lab

Summary: 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 103and 105 cells.ml-1, 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 103-104 cells.ml-1, 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. .justify:after { content: ""; display:inline-block; width: 100%; }

Affiliations: Northeastern University, Northeastern University, Northeastern University

Version: 1

Publication Date: 2019

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