TITLE: Environmental DNA (eDNA) extraction using Qiagen DNeasy Blood and Tissue Kit
AUTHORS: Kristine Walz, Kevan  Yamahara, Reiko Michisaki, Francisco Chavez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Nucleic acids extraction from the filters using the Qiagen DNeasy Blood and Tissue Kit with some modifications to the manufacturer’s protocol. These extraction protocols, adapted from Thomsen et al (2012), are used by the Monterey Bay Aquarium Research Institute (MBARI) for Monterey Bay, CA samples (MB). All MB samples were extracted in the Chavez Laboratory at the Monterey Bay Aquarium Research Institute, Moss Landing California.
 </div><div class = "text-block">This work was supported by NASA grant NNX14AP62A ‘National Marine Sanctuaries as Sentinel Sites for a Demonstration Marine Biodiversity Observation Network (MBON)’ funded under the National Ocean Partnership Program (NOPP RFP NOAA-NOS-IOOS-2014-2003803 in partnership between NOAA, BOEM, and NASA), and the U.S. Integrated Ocean Observing System (IOOS) Program Office.</div><div class = "text-block"><span>Thomsen, P. F., Kielgast, J., Iversen, L. L. N., Møller, P. R., Rasmussen, M. & Willerslev, E., 2012, Detection of a Diverse Marine Fish Fauna Using Environmental DNA from Seawater Samples. </span><span style = "font-style:italic;">Plos One,</span><span> 7(8)</span><span style = "font-weight:bold;">,</span><span>e41732.</span></div><div class = "text-block">Qiagen Inc., July 2006 DNeasy Blood and Tissue Handbook; available online from Qiagen Inc§ .</div><div class = "text-block">Qiagen, Inc., June 2012 Qiamp DNA Mini and Blood Mini Handbook, available online from Qiagen, Inc.</div></div>

[STEPS]
?. [Preparation]
Nucleic acids were extracted from the filters using the Qiagen DNeasy Blood and Tissue Kit with some modifications to the manufacturer’s protocol.
?. [Preparation]
Prior to extraction, 0.5 mm and 0.1 mm glass beads (BioSpec Products) were ashed at 500 °C for 5 hours.
?. [Preparation]
Bead tubes: 0.25 g of each size glass bead was distributed into sterile 2.0-ml conical microcentrifuge tubes (with screw cap and o-ring). The tubes were subsequently autoclaved for 30 min.
?. [Extraction]
Sample filters were transferred to bead tubes with sterile forceps, 720 μl Buffer ATL (Qiagen) was added, and two bead-beating steps were done: tubes were shaken in a MiniBeadbeater (BioSpec Products) at maximum speed for 45 sec, followed by incubation at 56 °C for 30 min and a second round of bead-beating (45 sec) and incubation (30 min). After the second incubation, 80 μl Proteinase K (Qiagen) was added. Tubes were then placed in a 56 °C shaking incubator for overnight incubation.
?. [Extraction]
To serve as a control in each set of extractions, an additional empty tube with beads was carried through the process as an extraction blank.
?. [Extraction]
After incubation, tubes were vortexed for 15 sec then centrifuged for 1 min at 4,000 x g. 650 μl of supernatant was transferred to new 1.5-ml tubes then spun at 13,000 x g for 1 min. After the final spin, 600 μl of supernatant (avoiding any remaining glass beads) was transferred to new 2-ml tubes for next steps.
?. [Extraction]
Remaining steps followed the manufacturer’s protocol for the Qiagen DNeasy Blood and Tissue Kit with the following modifications: 600 μl of Buffer AL and 600 μl of 100% ethanol were used; 500 μl of lysate was pipetted to spin column then pulled through with aQiagen vacuum manifold each time until the entire volume of lysate (1.8 mL) was pulled through the spin column; two 500-μl washes of Buffer AW1 and two 500-μl washes of Buffer AW2 were done; elutions were done in two 50-μl steps for a total of 100 μl extracted DNA.