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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.

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Version History
Version 0.1.1 Viewing Latest
Effective: 2026-04-20

Cross-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
Effective: 2026-04-20

Initial release. Salt-precipitation DNA extraction is a ~40-year-old field-standard technique; procedural facts uncopyrightable. Supports the eDNA arc downstream of procedure 78.

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Procedure Details
Safety & Hazards
  • 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.
Preparation Notes

Reagents:

  • Preserved filter (from procedure 79) — 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.

Timing
  • 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).
Equipment (Catalog) 5
  • 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
Materials (Catalog) 2
  • 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 0.1.1)

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 79), output Sample record with concentration annotation, and Assay record for the agarose gel QC if performed.

Completion Notes

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
  1. 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
  2. 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
  3. 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
  4. LibreBiotech procedure 79 — eDNA Water Filtration (upstream); procedure 72 — Standard PCR (downstream). Link protocol