SDS + Salt-Precipitation DNA Extraction (Consumer-Safe Cross-Tissue Protocol)

• BSL-1 work only. Food-grade or similarly unhazardous biological starting materials. Not validated for clinical, regulated, or infectious samples.
• No fume hood required. This is a deliberate design choice — the protocol substitutes high-salt protein precipitation for the chloroform phase-separation step used in procedure 82. Consumer kits built from this protocol can be used on a kitchen bench.
• SDS (sodium dodecyl sulfate). Cosmetic-grade detergent (used in shampoos and toothpaste). Mild skin/eye irritant as dry powder; cosmetic-grade exposure levels are well understood. Prepare stock solutions in a ventilated area to avoid inhaling dust.
• Proteinase K. Protease enzyme; wear gloves to prevent gradual skin degradation with repeated exposure.
• Isopropanol and ethanol. Flammable. Keep away from open flame during precipitation and wash steps.
• Saturated NaCl solution. Non-hazardous but dense and cold when chilled on ice; use closed tubes and inversion rather than open-vessel pours.
• Pipetting hygiene. Filter tips throughout to prevent aerosol carryover between samples — critical for the POC's cross-tissue comparison, and good practice for any extraction.
• Not a clinical extraction. Output is research-grade genomic DNA; not validated for diagnostic or forensic use.

Lysis buffer (make fresh, store at room temperature up to 1 month):

• 100 mM Tris-HCl pH 8.0
• 5 mM EDTA
• 0.2 M NaCl
• 0.5% SDS (w/v)
• Filter through a 0.22 µm filter or autoclave. Use a pH meter to adjust pH before filtering or autoclaving.

SDS precipitates at low temperatures — if the buffer has been stored cold and looks cloudy, warm to 37°C and mix on a magnetic stirrer briefly before use.

Saturated NaCl solution (shelf-stable, room temperature): ~6 M NaCl (~350 g NaCl in 1 L water with warming to dissolve). Allow to cool to room temperature before use. Some precipitation on cooling is expected and fine.

Proteinase K stock: 20 mg/mL in water; single-use aliquots at −20°C; never freeze-thaw.

Sample preparation per tissue type (captured in the tissue_type parameter on Process execution):

Mental model. SDS denatures proteins by coating them in negative charge, unfolding them, and solubilising them. Proteinase K cuts the denatured protein chains into small peptides. Adding a large excess of NaCl (final ~1.5 M) then causes the peptide-SDS complexes to aggregate and precipitate — they crash out of solution, but DNA stays in the supernatant because it's highly charged and hydrophilic. A quick centrifuge step pellets the protein/debris/SDS complex; the DNA-containing supernatant is transferred and precipitated with isopropanol as in any standard DNA prep. No organic solvent ever touches the sample — that's the core design feature.

Recipe lineage + honest deviations. The salting-out chemistry traces to Miller et al. 1988 (Nucleic Acids Research), which Qiagen later productised as the Puregene kit, which is the commercial template this protocol most closely resembles. The buffer numbers here (0.5% SDS, 100 mM Tris, 5 mM EDTA, 0.2 M initial NaCl, 0.2 mg/mL Proteinase K) deviate from both Miller 1988 (1% SDS, 10 mM Tris, 2 mM EDTA, 0.4 M NaCl, 0.1 mg/mL PK) and Aljanabi & Martinez 1997 (2% SDS, 10 mM Tris, 2 mM EDTA, 0.4 M NaCl, 40 µg/mL PK). They're a first-pass consumer-kit variant — internally consistent (lower SDS compensated by higher PK; lower initial NaCl because the salting-out step does the heavy lifting; higher Tris for better consumer-shelf pH stability), but not directly empirically tuned. The Stage 0.5 POC bench work will measure whether these numbers actually deliver across the five tissues, and v0.2.x forks the recipe if data justify.

What this gets you vs procedure 82 (CTAB). Procedure 82 gives higher yields and longer fragments (20-40+ kb) because CTAB + chloroform:IAA is a well-optimised HMW-DNA chemistry. This protocol typically delivers 5-20 kb fragments with ~50-70% of CTAB's yield — enough for most PCR amplicon work and for ONT library prep if the tissue starts clean. The trade is chemistry simplicity for some HMW performance. For the POC, we run both chemistries on the same tissues to measure the gap directly.

Why "consumer-safe" matters here. The whole point of this protocol is that everything in the reagent list can be legally purchased by a member of the public in Australia without an ABN (verified for the POC context as of 2026-04-21). SDS, NaCl, Tris buffer precursors, EDTA, isopropanol, and ethanol are all available through general-retail, pharmacy, or cosmetic-supplier channels. No chloroform (ABN-gated), no β-ME (ABN-gated), no liquid nitrogen (not consumer-accessible). This is the chemistry that survives the translation from Kevin's bench into a mailable kit.

• Tissue preparation: 5-15 min per sample (faster than CTAB because no LN₂ workflow).
• Lysis: 60 min at 55°C (extend to 120 min for hard seeds or dried mushrooms without bead beating).
• Salt precipitation + centrifuge: 15 min active (chill step can be done while other samples continue incubating).
• Precipitation + wash + drying + resuspension: 30 min.
• Total per batch: ~2 h active for 8 samples in parallel — marginally faster than CTAB because no fume-hood transfer is needed.