eDNA Extraction from Filter Membranes (Salt-Precipitation Method)
Salt-precipitation DNA extraction from water-filter membranes preserved in Longmire buffer. Recovers environmental DNA suitable for PCR, qPCR, and metabarcoding — without commercial spin columns, keeping the open-reagent ethos intact. Input: filter from procedure 78. Output: ~50 µL of DNA in TE buffer at 10–50 ng/µL. Cost: ~$0.20 per extraction (vs. $5–15 for commercial kits). Written for a novice audience.
Version History
Version 0.1.1 Latest
Effective: 2026-04-20Cross-reference fix: eDNA-filtration ref 78→79. All `procedure N` references converted to Markdown hyperlinks pointing at https://librebiotech.org/?action=show&id=N — enables in-app click-through to referenced sibling procedures. Text content otherwise preserved.
Version 0.1.0 Viewing
Effective: 2026-04-20Initial release. Salt-precipitation DNA extraction is a ~40-year-old field-standard technique; procedural facts uncopyrightable. Supports the eDNA arc downstream of procedure 78.
Procedure Details
- BSL-1 environmental samples. Treat filters as biologically active material; autoclave all waste.
- Proteinase K. Protease enzyme; standard gloves. Handle solutions with care.
- Chloroform (optional phase separation). TOXIC; carcinogen-suspect; strong irritant; volatile. Use ONLY in a fume hood with full PPE. This procedure uses a chloroform-free variant by default; chloroform version noted for reference only.
- Isopropanol / ethanol. Flammable; standard fume-hood or ventilated-area handling.
- High-salt solutions. Non-toxic but irritant; standard gloves.
- Contamination prevention. eDNA extractions are contamination-critical; dedicated pre-amp pipettes, fresh tips, clean benches.
Reagents:
- Preserved filter (from procedure 78) — in Longmire buffer.
- Proteinase K — 20 mg/mL stock at −20°C in single-use aliquots.
- 5 M NaCl — sterile-filtered; stable room temperature.
- 100% ethanol — molecular grade.
- 70% ethanol — prepared fresh in nuclease-free water.
- TE buffer — 10 mM Tris-HCl pH 8.0 + 0.1 mM EDTA. Or nuclease-free water.
Equipment:
- Heat block at 56°C.
- Microcentrifuge (≥13,000 × g).
- Sterile forceps.
- 1.5 mL and 15 mL microcentrifuge tubes.
- Ice bucket.
Mental model. Longmire-preserved filter contains captured DNA still embedded in cellular material. Proteinase K digestion at 56°C releases DNA into solution. High-salt NaCl addition destabilises DNA-protein interactions. Isopropanol precipitates DNA (and co-precipitates salts); 70% ethanol wash removes the salts. Resuspension in TE yields clean DNA ready for PCR.
Filter handling note: cellulose nitrate filters dissolve partially in Longmire + SDS, so some filter material enters the DNA solution. This is fine for PCR. GF/F glass-fibre filters do not dissolve; recovery is easier but bead-beating may be needed for strongly-bound DNA.
- Per sample: ~90 min active + overnight incubation (16–18 h).
- Batch: 12–24 samples in parallel with a heat block.
- Total elapsed: 2 days (overnight step dominates).
-
Microcentrifuge
Centrifugation
Specs: 13,000 × g, 4°C cooled ideally
For isopropanol precipitation -
Micropipette
Liquid handling
Specs: P20, P200, P1000; filter tips
Dedicated pre-amp -
Vortex mixer
Mixing
Specs: Standard benchtop
For brief mixing; never for DNA pellet -
Freezer (−20 °C)
Storage
Specs: Standard lab freezer
Final sample storage -
Heat block
Thermal regulation
Specs: 56°C capable for overnight use
Must hold 56°C accurately for 16–18 h
-
Proteinase K
Enzyme
Qty: 40 µL of 20 mg/mL per 800 µL Longmire
1 mg/mL final; NEB P8107S or equivalent -
Buffer
Reagent
Qty: 50 µL per sample
10 mM Tris-HCl pH 8.0 + 0.1 mM EDTA; or nuclease-free water
Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns
| Name | Type | Required | Default | Unit | Description |
|---|---|---|---|---|---|
proteinase_k_final_conc_mg_ml |
number | — |
1
|
— | Proteinase K final concentration during lysis. 1 mg/mL standard; scale up (2 mg/mL) for tough biomass. |
lysis_temp_c |
number | — |
56
|
degree Celsius (UO:0000027) | Temperature for Proteinase K digestion. 56°C standard; 50°C acceptable if heat block limited. |
lysis_time_h |
number | — |
17
|
hour (UO:0000032) | Duration of lysis. 16–18 h overnight standard. 4 h works but yields less DNA. |
nacl_ratio |
number | — |
0.5
|
— | Volume ratio of 5 M NaCl to lysate. 0.5× standard — final ~1.25 M NaCl in precipitation mix. |
isopropanol_ratio |
number | — |
1
|
— | Volume ratio of isopropanol to salted lysate. 1× standard. |
elution_volume_ul |
number | — |
50
|
microliter (UO:0000101) | Final elution volume. 50 µL standard; smaller volume (30 µL) more concentrated; larger (100 µL) more diluted. |
Procedure Steps (Version 0.1.0)
Retrieve the preserved filter tube(s) from −20°C storage. If tubes are frozen, thaw at room temperature for 10 minutes — do NOT warm above 37°C.
For 1.5 mL tubes (flat filters): pellet any sediment by brief centrifugation (30 s at 13,000 × g). For 15 mL conical tubes (Sterivex filters): skip this step.
Add 40 µL of 20 mg/mL Proteinase K to each sample tube (final concentration ~1 mg/mL in 800 µL Longmire). For Sterivex in 5 mL Longmire: add 250 µL Proteinase K.
Vortex briefly (5 s) to mix.
Incubate at 56°C overnight (16–18 h). This thoroughly lyses cellular material on the filter. Shorter incubation (4 h) may work but yields less.
The next day, cool tubes to room temperature.
If using a 1.5 mL flat-filter tube: remove the filter using sterile forceps, squeezing residual liquid back into the tube before disposal. If using a 15 mL Sterivex tube: pipette the supernatant directly from the tube, avoiding the cartridge.
Transfer the supernatant (now containing lysed filter + DNA + proteins + digestion products) to a new 2 mL tube. For Sterivex samples: transfer ~4 mL to a new 5 mL tube (or split across two 2 mL tubes).
Add 0.5 volume of 5 M NaCl (e.g. 400 µL to an 800 µL lysate). Invert 5–6 times to mix. The high salt concentration destabilises DNA-protein interactions.
Add 1 volume of 100% isopropanol (e.g. 1.2 mL to 1.2 mL salted lysate). Invert 5–6 times; the solution may become cloudy — this is DNA precipitate.
Incubate at room temperature for 10 minutes (some protocols use 4°C for 30 min — marginal improvement in yield but longer).
Centrifuge at 13,000 × g for 15 minutes at 4°C. A white pellet should be visible at the bottom of the tube.
Carefully discard the supernatant by pipetting or gentle inversion (the pellet may come loose — if in doubt, pipette from the top).
Add 500 µL of 70% ethanol to wash the pellet. Do not resuspend; just add gently.
Centrifuge at 13,000 × g for 5 minutes. Discard the ethanol.
Briefly spin the tube (30 s at low speed) and remove residual ethanol with a P20 pipette tip.
Air-dry the pellet for 5–10 minutes at room temperature with the tube open. Watch for the pellet becoming transparent (fully dry). Do not over-dry.
Resuspend the pellet in 50 µL TE buffer or nuclease-free water. Pipette up and down 10 times; vortex briefly.
Incubate at 37°C for 10 minutes with occasional mixing to fully dissolve.
Measure concentration on a Nanodrop or Qubit — note that eDNA extracts give noisy readings (see Completion Notes). Record the concentration as a Sample annotation in LibreBiotech.
Store at −20°C until downstream PCR/qPCR. Typical shelf life 6–12 months.
Record the extraction in LibreBiotech: Process record with procedure_version_id=80, input Sample record linking to the filter (from procedure 78), output Sample record with concentration annotation, and Assay record for the agarose gel QC if performed.
Expected outcome. ~50 µL of DNA in TE buffer. Concentration typically 10–50 ng/µL for a 1 L moderate-biomass water sample; higher for biomass-rich water, lower for oligotrophic. A260/A280 ratio 1.6–1.9 (slightly lower than gDNA due to filter dissolution).
QC. Run 2 µL on a 1% agarose gel (procedure 73). Expected: faint smear or light band, usually 500 bp – 5 kb range. Pure genomic DNA bands are rare in eDNA — the signal is usually a smear representing degraded environmental material.
Nanodrop caution: eDNA extracts often show noisy A260/A280 ratios due to filter-membrane dissolution contaminants. Confidence from Nanodrop alone is low; proceed to downstream PCR / qPCR with a positive-control spike to confirm extraction worked.
Storage. −20°C for months; −80°C for years. Avoid repeated freeze-thaw (<5 cycles).
Downstream. PCR with eDNA-compatible primers (mini-barcoding for fish, COI arthropod primers, bacterial 16S, etc.) via procedure 72. Species call via procedure 76.
Troubleshooting.
| Symptom | Likely cause | Fix |
|---|---|---|
| No visible DNA pellet after isopropanol precipitation | Very low biomass; degraded DNA | Proceed to PCR anyway — low-concentration eDNA may still amplify; increase template volume in PCR |
| Low A260/A280 ratio (<1.6) | Filter-membrane dissolution carryover | Acceptable for eDNA; continue to PCR; consider additional cleanup (SPRI procedure 74) if PCR inhibition observed |
| PCR inhibition (no amplification even with positive control spike) | Humic acids, other inhibitors | Dilute extract 1:5 or 1:10 and retry; or SPRI cleanup first |
| Very high A260/A280 (>2.0) | RNA contamination | Add RNase A treatment (20 µg/mL, 37°C × 30 min) before salt precipitation |
| Gel shows clean genomic DNA band | Unusual for eDNA — suggests bulk-tissue contamination | Check sampling protocol; was a tissue-carrying animal handled during collection? |
References
- Miller SA, Dykes DD, Polesky HF (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16(3):1215. (Foundational salt-precipitation method). DOI paper
- Deiner K, Walser JC, Mächler E, Altermatt F (2015). Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA. Biol Conserv 183:53–63. DOI paper
- Renshaw MA, Olds BP, Jerde CL, McVeigh MM, Lodge DM (2015). The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNA extraction. Mol Ecol Resour 15(1):168–76. DOI paper
- LibreBiotech procedure 78 — eDNA Water Filtration (upstream); procedure 72 — Standard PCR (downstream). Link protocol