bd1. Water
Collection
- Grab sampling: collect a fixed volume (for example 5–8 L) into a sterile, DNA-free bottle. Use a depth sampler (for example Van Dorn) for deeper water.
- Filtration (preferred): filter as soon as possible through a 0.22–0.45 µm membrane using a peristaltic pump, hand vacuum pump or syringe filter. Remove the filter with sterile forceps into a sterile 2 mL tube.
- No filtration available: freeze the water directly at −20 to −80 °C.
Preservation
Keep filters in Longmire’s buffer, ethanol or RNAlater in the field, or freeze at −20 to −80 °C.
| Problem | Cause | Solution |
|---|---|---|
| Contamination | Equipment, bottles, human DNA | Gloves, sterile disposables, field blanks |
| DNA degradation | Sunlight, heat, delay | Filter quickly; keep cold or in buffer |
| Clogged filters | Turbid water, algae | Pre-filter, use several filters or less volume |
| Low DNA yield | Dilute or old samples | Larger volume; replicate samples |
2. Soil and sediment
- Wear sterile gloves and use clean, sterile scoops. Remove surface debris (leaves) and collect about 5–10 g from 0–10 cm depth.
- Put into sterile tubes or Whirl-Pak bags. In variable sites, take several sub-samples and pool them.
- Freeze at −20 or −80 °C as soon as possible, or use a soil preservation solution or silica gel in the field.
| Problem | Cause | Solution |
|---|---|---|
| PCR inhibitors | Humic acids, organic matter | Inhibitor-removal extraction kits |
| Cross-contamination | Shared tools between sites | Disposable or sterilised tools |
| Uneven results | Soil is patchy | Composite samples and replicates |
| Degraded DNA | Heat, microbial activity | Keep cool; process quickly |
3. Air (advanced)
- Use an air sampler with HEPA or PTFE filters; volume depends on flow rate and time (for example 30 L/min for 30 minutes).
- Store filters on dry ice or in preservative, then at −80 °C. Wear full protection (mask, gloves, hair net): airborne DNA is very low and easily contaminated.
4. Good practice for all eDNA samples
- Controls: include a field or filtration blank (sterile water handled like a sample).
- Change gloves between samples; sterilise reusable equipment with bleach or ethanol, or use disposables.
- Replicates and records: collect replicates and note GPS, weather, time, temperature, pH and turbidity.
- Transport: keep on ice or in a cool box until frozen; avoid freeze–thaw.
| Source | Method | Amount | Storage |
|---|---|---|---|
| Water | Filtration, 0.22–0.45 µm | 0.5–5 L | −20 °C or buffer |
| Soil | Sterile scoop, tube | 5–10 g | −80 °C or buffer |
| Sediment | Core or scoop | 10 g or more | −80 °C or buffer |
| Air | HEPA / PTFE filter | Depends on airflow | −80 °C |