Introduction

Before collecting a single sample, it’s important to think through the full design of your eDNA project. This section introduces the considerations and protocols for preparing and collecting high-quality eDNA samples in both field and museum environments.

Designing an eDNA Study

What’s the research question?

  • Are you detecting a specific species or surveying communities?
  • Temporal and spatial scales matter.

Choosing the right marker

  • Based on your taxa of interest (e.g., COI for animals, 18S for eukaryotes).
  • Consider ecosystem and sample type (water vs. sediment vs. swabs).

Consider DNA persistence

  • Environmental DNA degrades over time.
  • Degradation influenced by UV light, temperature, pH, microbial activity.

Sampling Environments

Aquatic Sampling

  • Surface water, mid-column, sediment cores.
  • Marine and freshwater protocols differ in filtration volume and salinity handling.

Terrestrial and Air Sampling

  • Soil or snow sampling using sterile scoops or syringes.
  • Air filters or cyclone samplers for airborne DNA.

Museum Settings

  • Swabbing collection surfaces, containers, or tools.
  • Extracting DNA from ethanol or residual fluids.

Filtration and Preservation

Filter Types

  • Sterivex (enclosed, on-site filtration)
  • Glass fiber or cellulose nitrate filters (lab filtration)

Volume Considerations

  • 250 mL to 2 L common for water samples
  • Use peristaltic pumps or syringes with luer-lock filter holders

Preservation Options

  • Ethanol or Longmire’s buffer
  • Dry preservation (silica desiccant)
  • Freeze at -20°C or lower ASAP

Contamination Control

Field Protocols

  • Wear gloves and change them between samples
  • Sterilize gear with 10% bleach + rinse
  • Use single-use consumables when possible

Controls

  • Field blanks (filtered water from the field site)
  • Filtration blanks (no sample passed through filter)
  • Lab blanks (control throughout extraction process)

Metadata Collection

Essential Fields

  • GPS coordinates
  • Date and time
  • Water temperature, salinity, turbidity, pH
  • Weather and tides (for marine)

Museum Context

  • Specimen ID and accession numbers
  • Container type, date of preservation
  • Preservation fluid composition

Field Kits and Logistics

  • Pre-label tubes and filters
  • Bring extra gloves, sample tubes, waste containers
  • Clean workspace for filtering (e.g., in the car, pop-up lab tent)

Preservation Challenges in Museum Samples

Ethanol Preservation

  • Ethanol can preserve DNA fairly well, especially at high concentrations (95–100%).
  • However, over time, ethanol can cause hydrolysis, leading to DNA fragmentation.
  • Repeated ethanol changes or evaporative concentration can alter preservation effectiveness.
  • DNA yield is typically lower in aged ethanol than fresh environmental samples.

Formalin Fixation

  • Formalin crosslinks proteins and nucleic acids, severely degrading or blocking PCR amplification.
  • DNA in formalin-fixed samples is often:
    • Highly fragmented
    • Chemically modified (e.g., methylol adducts)
    • Difficult to extract without specialized protocols (e.g., reversal treatments or harsh lysis)

Considerations for Museum Researchers

  • Prioritize ethanol-preserved materials when possible
  • Use extraction protocols designed for low-yield or fragmented DNA
  • Document preservative type and history in metadata

Foundational Reading

Summary

Good eDNA starts with good planning. Clear protocols, contamination controls, and rich metadata ensure high-quality results and reproducibility.