Jan 29, 2019

Public workspaceMBARI Environmental DNA (eDNA) extraction using Qiagen DNeasy Blood and Tissue Kit

DOI
dx.doi.org/10.17504/protocols.io.xjufknw
  • 1Monterey Bay Aquarium Research Institute, Moss Landing, CA;
  • 2Monterey Bay Aquarium Research Institute
  • MBON eDNA
Reiko P Michisaki
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.xjufknw
Protocol Citation: Kristine Walz, Kevan Yamahara, Reiko P Michisaki, Francisco P. Chavez 2019. MBARI Environmental DNA (eDNA) extraction using Qiagen DNeasy Blood and Tissue Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.xjufknw
Manuscript citation:
Kristine Walz, Kevan Yamahara, Reiko P Michisaki, Francisco P. Chavez. MBARI Environmental DNA (eDNA) extraction using Qiagen DNeasy Blood and Tissue Kit (2019) Monterey Bay Aquarium Research Institute, Moss Landing, CA, dx.doi.org/10.17504/protocols.io.xjufknw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 29, 2019
Last Modified: January 29, 2019
Protocol Integer ID: 19796
Keywords: environmental DNA, marine, extraction, MBARI, Monterey Bay Aquarium Research Institute, MBON, Marine Biodiversity Observation Network, Monterey Bay California, seawater extraction
Abstract
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.
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.

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. Plos One, 7(8),e41732.

Qiagen Inc., July 2006 DNeasy Blood and Tissue Handbook; available online from Qiagen Inc§ .
Qiagen, Inc., June 2012 Qiamp DNA Mini and Blood Mini Handbook, available online from Qiagen, Inc.
Guidelines
Always observe proper laboratory safety warning and precautions.
Safety warnings
This section is taken directly from the Qiagen DNeasy Blood and Tissue Handbook.
  • When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs). These are available online in convenient and compact PDF format at www.qiagen.com/ts/msds.asp where you can find, view, and print the MSDS for each QIAGEN kit and kit component.
  • CAUTION: DO NOT add bleach or acidic solutions directly to the sample-preparation waste.
  • Buffer AL and Buffer AW1 contain guanidine hydrochloride, which can form highly reactive compounds when combined with bleach. If liquid containing this buffer is spilt, clean with suitable laboratory detergent and water. If the spilt liquid contains potentially infectious agents, clean the affected area first with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite.
  • The following risk and safety phrases apply to components of DNeasy Blood & Tissue Kits and DNeasy 96 Blood & Tissue Kits.
  • Buffer AL and Buffer AW1 (concentrate)Contains guanidine hydrochloride: harmful, irritant. Risk and safety phrases:* R22-36/38, S13-26-36-46
  • 1 Proteinase K  Contains proteinase K: sensitizer, irritant. Risk and safety phrases:* R36/37/38-42/43, S23-24-26-36/37 13.10 24-hour emergency information
  • Emergency medical information in English, French, and German can be obtained 24 hours a day from: Poison Information Center Mainz, Germany Tel: +49-6131-19240; or Poison Control USA Tel: (800) 222-1222.
  • R22: Harmful if swallowed;
  • R36/37/38: Irritating to eyes, respiratory system and skin;
  • R36/38: Irritating to eyes and skin;
  • R42/43: May cause sensitization by inhalation and skin contact;
  • S13: Keep away from food, drink, and animal feeding stuffs;
  • S23: Do not breathe spray;
  • S24: Avoid contact with skin;
  • S26: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice;
  • S36: Wear suitable protective clothing;
  • S36/37: Wear suitable protective clothing and gloves;
  • S46: If swallowed, seek medical advice immediately, and show container or label
Preparation
Preparation
Nucleic acids were extracted from the filters using the Qiagen DNeasy Blood and Tissue Kit with some modifications to the manufacturer’s protocol.
Prior to extraction, 0.5 mm and 0.1 mm glass beads (BioSpec Products) were ashed at 500 °C for 5 hours.
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
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.
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.
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.
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.