Jun 13, 2024

Public workspaceAutomated eDNA Extraction from Estuarine Samples Using Magnetic Beads

DOI
dx.doi.org/10.17504/protocols.io.5jyl82jn9l2w/v1
Automated eDNA Extraction from Estuarine Samples Using Magnetic Beads
  • Fouad El Baidouri 1,
  • Muriel Kelly2,
  • Heather L. Gilbert1,
  • Alison Watts1
  • 1Department of Civil & Environmental Engineering, University of New Hampshire, Durham, NH, USA.;
  • 2Hubbard Center for Genome Studies, University of New Hampshire, Durham, NH, USA
Fouad El Baidouri
Open access
DOI: dx.doi.org/10.17504/protocols.io.5jyl82jn9l2w/v1
Protocol Citation: Fouad El Baidouri , Muriel Kelly, Heather L. Gilbert, Alison Watts 2024. Automated eDNA Extraction from Estuarine Samples Using Magnetic Beads. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82jn9l2w/v1
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: May 13, 2024
Last Modified: June 13, 2024
Protocol Integer ID: 99676
Keywords: eDNA, Environmental DNA, Automation, PCR inhibitor removal, DNA extraction, Magnetic beads, Environmental biomonitoring, Automated extraction, Zymo clean up, KingFisher instrument
Abstract
This method presents an optimized protocol for the automated extraction of environmental DNA (eDNA) from estuarine samples using magnetic beads. The addition of Proteinase K to the lysis solution facilitates DNA solubilization at 56°C and mechanical rotation ensures thorough lysis. The DNA is then purified using a double ethanol wash and magnetic bead-based extraction on a KingFisher system. The method is designed for filters preserved in Longmire's buffer. If a different preservation is used the first step should be modified.
Guidelines
Follow Personal Protective Equipment (PPE) procedures.
  • Clean workspace: Thoroughly clean all work surfaces including the benchtop and equipment exteriors using a 5%-10% bleach solution followed by a distilled water rinse. Wipe down pipettes, pipette tip boxes, sharpies, pens and any other tools that will be used during the protocol to avoid cross contamination.
  • Incubator check: Verify that the incubator is operational and set to the correct temperature before use (56°C ).
  • Handling: Use aseptic techniques to avoid introducing contaminants into sample preparations. Include extraction blanks to check for cross contamination from adjacent wells.
  • Storage: If immediate DNA extraction is not possible, samples can be stored in Longmire's buffer at room temperature for up to 50 days (minimize exposure to light), or frozen at -20°C or -80°C for long-term storage.

Notes

  • Illumina Tune beads (CAT: 20060057) performed better than Ampure XP and are cheaper.
Materials
Protective gear
  • Gloves: to eliminate/reduce contamination when handling samples
  • Lab coat

Cleaning supplies
  • 5%-10% bleach solution: for cleaning benches and equipment
  • Distilled water: for rinsing

Consumables
  • Deep Well Plates (DWP) (97002540)
  • 200 µl plates: for elution (97002540)
  • Tip comb for DW magnets (97002534)
  • Pipettes and pipette tips: 1,000 μl, 200 μl, 20 μl
  • Reagent troughs/reservoirs
  • Tape: for securing during incubation

Reagents
  • Longmire’s buffer (see References): prepare using the following components to make a total of 500 mL:
   -50 mL of 1 M Tris-HCl, pH 8.0 (for buffering the solution)
   -100 mL of 0.5 M EDTA, pH 8.0 (chelates divalent cations and prevents degradation by nucleases)
   -1 mL of 5 M NaCl (provides ionic strength to the buffer)
   -12.5 mL of 20% SDS (w/v) (a detergent that aids in cell lysis and protein solubilizing)
   -336.5 mL of PCR grade water (used to bring the solution up to the final volume and ensure purity)
  • Proteinase K: from Promega (CAT: PRMC5005)
  • 80% ethanol: used for wash steps
  • Tris-HCl (10 mM): used in the elution plate
  • Illumina Tune Beads (CAT: 20060057): for magnetic bead based DNA extraction

Equipment
Incubator with a rotator: set at 56°C for incubation
Multichannel pipette (i.e. Integra): for efficient handling of multiple samples
Kingfisher Flex Purification System
Qubit reagents or HTS Microplate Reader: for quantifying DNA concentration
Nanodrop analyzer: for assessing DNA quality (optional)
 
Safety warnings
Attention
Ensure all surfaces and equipment are thoroughly cleaned with 5%-10% bleach and rinsed with distilled water to prevent contamination. Include extraction blanks to check for sample cross contamination.
Before start
Longmire’s buffer preparation: ensure that Longmire's buffer is prepared using PCR grade water.
Sample Preparation
Sample Preparation
Perform the following steps in a biosafety cabinet under aseptic conditions
For samples collected on filters stored in Longmire's buffer (wet filters)
  • Pipette out all of the Longmire's buffer from the sample tube into a separate Eppendorf tube (leave the sample filter in the sample tube)
  • Pipette 490 μl per filter of the original Longmire's buffer back into the sample tube
  • Add 10 μl of Proteinase K per filter into the sample tube (total volume 500 μl)
  • Using the pipette tip, mix the reagents thoroughly
  • Using the same pipette tip, ensure the filter(s) are completely submerged in the Longmire's buffer/Proteinase K solution
For dry filters, add 1000 μl of Longmire's solution to the tube, ensuring the filter is completely submerged - Incubate for 90 min to overnight.
  • Pipette out all of the Longmire's buffer from the sample tube into a separate Eppendorf tube (leave the sample filter in the sample tube)
  • Pipette 490 μl per filter of the original Longmire's buffer back into the sample tube
  • Add 10 μl of Proteinase K per filter into the sample tube (total volume 500 μl)
  • Using the pipette tip, mix the reagents thoroughly
  • Using the same pipette tip, ensure the filter(s) are completely submerged in the Longmire's buffer/Proteinase K solution
Sample Incubation & Lysis
Sample Incubation & Lysis
  • Place tubes in tube rack on a rotating or gently shaking surface in a 56°C incubator
  • Secure rack with tape
  • Turn on rotator
  • Incubate for 90 minutes or overnight
  • After incubation proceed with steps below. Alternatively samples can be kept at 4°C overnight for a later extraction.
Incubation
Washing and Elution Plates Preparation
  • During the 90 minute incubation step, prepare two wash plates by pipetting 500 μl freshly prepared 80% ethanol into the wells of two DWPs
  • Prepare an elution plate by pipetting 100 μL of 10 mM Tris-HCl into the wells of a 200 μl plate
Magnetic Beads Addition
Magnetic Beads Addition
  • After incubation, transfer 400 μl of the supernatant lysis to a new DWP (this is the sample plate). Bubble formation sometimes occurs due to the presence of SDS. Ensure the tubes are tightly sealed to prevent evaporation during the 56°C incubation period
  • Add 320 μl of Illumina Tune beads to the sample plate wells (1:8 sample to beads ratio to remove small DNA fragments < 200bp)
KingFisher Extraction (DNA Purification)
KingFisher Extraction (DNA Purification)
  • Place the sample plate (containing the 720 μl of sample + beads), wash plates 1 and 2 and the elution plate into the KingFisher and run the program as follows
  • The KingFisher is optimized for the volumes stated in this protocol
KingFisher Program

1. Pick up:
  •    Tip comb plate
2. Binding:
  •    Mix for 5 minutes at medium speed
  •    Collect beads, count: 5
  •    Move to sample plate
3. 1st Wash:
  •    Release beads
  •    Bottom mix and medium speed, each for 10 seconds, loop count 2
  •    Collect beads, count: 3; collect time: 5 seconds
  •    Transfer to wash plate 1
4. 2nd Wash:
  •    Release beads for 20 seconds
  •    Bottom mix and medium speed, each for 10 seconds, loop count 2
  •    Collect beads, count: 4; collect time: 5 seconds
  •    Transfer to wash plate 2
5. Dry:
  •    Use wash plate 2
6. Elution:
  •    Preheat to 60°C
  •    Bottom mix for 15 seconds, medium speed for 45 seconds, loop count: 6
  •    Collect beads, count: 1
  •    Transfer to elution plate
7. Collection of Beads:
  •    2 minutes at slow speed
  • After the program ends, immediately proceed with the Zymo cleanup steps using the elution plate, or store the plate at -20°C for future use. Dispose of chemical reagents according to Lab Hazardous Waste Management Plan.
Protocol references
Sanches, T.M. and Schreier, A.D., 2020. Optimizing an eDNA protocol for estuarine environments: Balancing sensitivity, cost and time. PLoS One, 15(5), p.e0233522.

Longmire, J.L., Maltbie, M. and Baker, R.J., 1997. Use of" lysis buffer" in DNA isolation and its implication for museum collections.