SDS + Salt-Precipitation DNA Extraction (Consumer-Safe Cross-Tissue Protocol)
Genomic DNA extraction using SDS lysis, high-salt protein precipitation, and isopropanol precipitation. No chloroform, no β-mercaptoethanol, no liquid nitrogen — every reagent is consumer-accessible in Australia without ABN gating, and every step can be executed with kitchen-or-classroom-scale equipment. Designed as the consumer-kit-compatible chemistry for the Stage 0.5 Cross-Tissue Extraction Feasibility POC, paired against procedure 82 (CTAB reference) across honey (pollen pellet from procedure 84), dried insects, plant seeds, dried mushrooms, and fish tissue. Produces DNA suitable for fluorometric quantification, agarose gel QC, and ONT library prep where fragment size permits. Written for a novice audience.
Version History
Version 0.1.2 Latest
Effective: 2026-05-18Tagged 9 process-scoped extraction-condition parameters with scope='process' to leverage spec-layer scope distinction (sibling bump to procedure 82 v0.1.2). Sample-scoped params (tissue_type, tissue_mass_mg, mechanical_disruption_method) unchanged. mechanical_disruption_method stays sample-scoped because different sample types require different physical prep (no LN2 in consumer-safe protocol). Bridge change only; protocol body identical to v0.1.1.
Version 0.1.1
Effective: 2026-04-21Dev review 2026-04-21 folded in: (1) recipe framing honestly labelled as Miller/Puregene-style variant; buffer-number deviations from Miller 1988 + Aljanabi 1997 primary references acknowledged in preparation notes + docstring. (2) Rotator mixer (equipment_types id 7) added for gentle resuspension. (3) Refrigerator 4 °C (equipment_types id 20) added for the ice-chill step + short-term extract storage. (4) Forward-reference IDs corrected: self-references to 'procedure 83' (now procedure 84 — pollen recovery) fixed; Qubit 84→85. (5) Bare `procedure N` references converted to Markdown hyperlinks. Text content otherwise preserved from v0.1.0 except for the explicit changes above.
Version 0.1.0 Viewing
Effective: 2026-04-21Initial release. Consumer-kit-compatible extraction chemistry for the Stage 0.5 POC, paired against procedure 82 (CTAB lab-grade reference). Miller/Puregene-style salting-out method (Miller et al. 1988; ~37 years old, field-standard). Unified protocol parameterised across five tissue types; no organic solvents, no cryogens, no ABN-gated reagents.
Procedure Details
- 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):
| Tissue | Mass input | Preparation | Notes |
|---|---|---|---|
| Pollen pellet (from honey, procedure 83) | Full pellet | Resuspend pellet in 300 µL lysis buffer before continuing | No grinding; 65°C heat-step opens pollen walls |
| Dried insects | 10-50 mg whole body | Crush in tube with a clean plastic pestle, or bead-beat 30 s at 5,000 RPM | Chitin fragments at lysis; full homogenisation not required |
| Plant seeds | 50-100 mg | Crack with mortar and pestle (no LN₂ needed) or bead-beat 60 s at 5,000 RPM | Without LN₂, extend lysis to 120 min at 55°C to compensate for less complete tissue fragmentation |
| Dried mushrooms | 50 mg | Cut small pieces with a clean blade; optional rehydration in buffer 5 min | Fungal chitin softens quickly once hydrated |
| Fish tissue | 20-50 mg | Mince with a clean blade | Soft tissue; minimum mechanical prep needed |
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.
What this gets you vs procedure 82 (CTAB). Procedure 81 gives higher yields and longer fragments (20-40+ kb) because CTAB + chloroform 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.
-
Balance / analytical scale
Specs: Two-decimal mg accuracy for tissue weighing
Tare to the sample tube; record mass as process parameter -
Bead beater / homogeniser
Specs: 1.5 mL tube format, 5,000 RPM capable
Mechanical disruption — primary tissue-prep method for seeds, insects, mushrooms -
Mortar and pestle
Specs: Ceramic, no cooling required
Alternative to bead beater for seeds; no LN₂ needed in this protocol -
Microcentrifuge
Centrifugation
Specs: ≥12,000 × g; room temperature capable
Main workhorse -
Micropipette
Liquid handling
Specs: P20, P200, P1000; filter tips throughout
Wide-bore or cut tips for aqueous phase transfer after salt precip -
pH meter
Measurement
Specs: Benchtop or pocket; calibrate with pH 4/7/10
Lysis buffer pH adjustment during preparation -
Spectrophotometer
Measurement
Specs: UV-capable (A260/A230/A280); optional
NanoDrop-style for purity QC after extraction -
Magnetic stirrer
Mixing
Specs: Ambient or gently warmed
Lysis buffer and saturated-NaCl preparation -
Vortex mixer
Mixing
Specs: Standard benchtop
Used for buffer preparation and the vigorous mix-after-salt step; NOT for DNA during or after the isopropanol precipitation -
Heat block
Thermal regulation
Specs: 55°C + 37°C capable
Lysis at 55°C; resuspension option at 37°C -
Water bath
Thermal regulation
Specs: 55°C capable (alternative to heat block)
For batches > 12 samples where heat block slots are full
-
EDTA
Chemical
Qty: 3 µmol
5 mM in lysis buffer -
Ethanol
Chemical
Qty: 1000 µL
70% v/v, freshly diluted; 500 µL per wash × 1-2 washes -
Isopropanol
Chemical
Qty: 420 µL
0.7 volumes of recovered supernatant (~600 µL); room temperature -
NaCl
Chemical
Qty: 1320 µmol
0.2 M (120 µmol) in lysis buffer + 1200 µmol in the 200 µL saturated-NaCl precipitation step -
SDS
Chemical
Qty: 3 mg
0.5% w/v in 600 µL lysis buffer; cosmetic-grade acceptable -
TE buffer
Chemical
Qty: 50 µL
10 mM Tris-HCl pH 8.0, 1 mM EDTA; resuspension volume -
Tris-HCl
Chemical
Qty: 60 µmol
100 mM in lysis buffer at pH 8.0 -
Filter pipette tips
Consumable
Qty: 6 tips
Filtered tips for supernatant transfer after salt precipitation and for final resuspension handling -
Microcentrifuge tube
Consumable
Qty: 2 tubes
1.5 mL or 2.0 mL per sample; one for lysis/extraction, one for final resuspension -
Pipette tips
Consumable
Qty: 8 tips
Generic tips for non-DNA-handling pipetting -
Proteinase K
Enzyme
Qty: 6 µL of 20 mg/mL
NEB P8107S or similar; single-use aliquots at −20°C
Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns
| Name | Type | Required | Default | Unit | Description |
|---|---|---|---|---|---|
tissue_type |
text | — | — | Starting material type. Valid values: pollen_pellet, insect_dried, plant_seed, mushroom_dried, fish_tissue. Controls tissue-preparation step in practice. | |
tissue_mass_mg |
number |
50
|
— | Tissue input mass in milligrams. Adjust per tissue: 10-50 for insects, 50-100 for seeds, 50 for mushrooms/fish, pellet-dependent for pollen. | |
lysis_buffer_volume_ul |
number | — |
600
|
— | Volume of SDS-Tris-EDTA-NaCl lysis buffer added per sample, in µL. 600 µL standard. |
lysis_temp_c |
number | — |
55
|
degree Celsius (UO:0000027) | Lysis incubation temperature in °C. 55°C optimal for SDS + Proteinase K activity; lower than CTAB's 65°C. |
lysis_time_min |
number | — |
60
|
minute (UO:0000031) | Lysis incubation time in minutes. 60 min standard; extend to 90-120 min for recalcitrant tissues (hard seeds without bead-beating, dried mushrooms). |
proteinase_k_final_mg_ml |
number | — |
0.2
|
— | Final Proteinase K concentration in the lysis tube, in mg/mL. 0.2 mg/mL standard — higher than CTAB because SDS lysis produces more denatured protein to digest. |
salt_precip_volume_ul |
number | — |
200
|
— | Volume of saturated (~6 M) NaCl added for protein salting-out, in µL. 200 µL standard (1/3 of lysis volume); final ~1.5 M NaCl after mixing. |
ice_chill_min |
number | — |
10
|
minute (UO:0000031) | Duration of ice chill after salt addition, in minutes. 10 min standard; promotes protein-SDS aggregate pelletability. |
isopropanol_volume_ul |
number | — |
420
|
— | Volume of isopropanol for DNA precipitation, in µL. 0.7 × recovered supernatant (~600 µL) = 420 µL standard. |
ethanol_wash_cycles |
number | — |
1
|
— | Number of 70% ethanol wash cycles. 1 standard for clean tissues; 2 for polyphenol/polysaccharide-heavy tissues (plant seeds, mushrooms). |
resuspension_volume_ul |
number | — |
50
|
— | Volume of TE buffer for final resuspension, in µL. 50 µL standard; scale down (25-30 µL) for low-yield substrates to concentrate. |
mechanical_disruption_method |
text | — |
bead_beat
|
— | Tissue disruption method before lysis. Valid values: bead_beat (preferred, 30-60s at 5000 RPM), mortar_pestle (no LN₂), blade_mince (soft tissue only), none (pollen pellet). |
Procedure Steps (Version 0.1.0)
Prepare tissue according to the tissue_type parameter (see preparation notes). Weigh or measure into a labelled 1.5 mL or 2.0 mL microcentrifuge tube. No liquid nitrogen is used in this protocol — mechanical disruption (pestle, bead beater, or blade) is sufficient across all supported tissues.
Record sample metadata in LibreBiotech: tissue type, starting mass, organism (as organism_curie), sample provenance, and any tissue-specific notes as Sample annotations. This creates the ISA-canonical origin record.
Pre-warm lysis buffer to 55°C in a heat block or water bath. If the buffer has been stored cold and looks cloudy, warm and mix first — SDS precipitates when cold.
Add 600 µL of pre-warmed lysis buffer to each sample tube. Mix gently by inverting 5× or tapping; do not vortex yet (HMW DNA shears under mechanical agitation).
Add 6 µL of 20 mg/mL Proteinase K stock (final concentration ~0.2 mg/mL). Mix gently by inversion. Proteinase K concentration is higher here than in procedure 82 because the SDS-detergent lysis needs stronger protein digestion to free all DNA.
Incubate at 55°C for 60 minutes in a heat block or water bath. Invert tubes gently every 15 minutes to keep tissue suspended. For recalcitrant tissues (hard seeds without bead-beating, dried mushrooms), extend to 120 minutes.
Cool tubes to room temperature (2-5 minutes on bench). Do not chill — SDS precipitates below 15°C.
Add 200 µL of saturated NaCl solution (~6 M) to each tube (approximately 1/3 the lysis volume). Cap firmly and shake vigorously for 20 seconds — unlike the CTAB chloroform step, vigorous mixing here is intentional. The tube contents should turn cloudy with precipitated protein/SDS complexes.
Chill tubes on ice for 10 minutes. The cold plus high salt maximises protein aggregation into a pelletable form.
Centrifuge at 12,000 × g for 10 minutes at room temperature (or 4°C if available). A tight white/cloudy pellet should form at the bottom; the supernatant above it is the DNA-containing aqueous phase.
Carefully transfer ~600 µL of the supernatant to a new labelled 1.5 mL microcentrifuge tube using a filter pipette tip. Tilt the tube and pipette from the top down — avoid disturbing the protein pellet at the bottom.
Add 0.7 volumes (typically 420 µL) of room-temperature isopropanol to the supernatant. Mix by gentle inversion for 30 seconds. Incubate at room temperature for 5 minutes; no benefit to extending further.
Centrifuge at 12,000 × g for 10 minutes at room temperature. A white or translucent pellet should form. Remove supernatant by pipetting; do not pour off.
Wash the pellet by adding 500 µL of freshly-diluted 70% ethanol. Do not mix or vortex — the wash is surface desalting. Centrifuge at 12,000 × g for 5 minutes. Remove supernatant carefully with a pipette.
(Optional for high-polyphenol or polysaccharide-heavy tissues — seeds, mushrooms.) Repeat the 70% ethanol wash once more.
Air-dry the pellet at room temperature for 5-10 minutes, or in a 37°C heat block for 2-3 minutes. Do not over-dry — over-dried HMW DNA is very hard to resuspend. The pellet should look just-dry, not chalky.
Resuspend the pellet in 50 µL of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Incubate at 4°C overnight, or at 37°C for 30 minutes with gentle flicking every 10 minutes, to fully resuspend. Pipetting up and down will shear the DNA; use flicks or tube rotation instead.
Record the extraction as a Process in LibreBiotech, linking input Sample → output Sample via source_samples lineage. Record all tissue-specific parameters (mass, lysis time, salt volume) as process_actuals. Output sample material_type should be extract.
Quantify DNA via procedure 84 (Qubit fluorometric dsDNA quant — to be submitted alongside this one) and assess fragment size on procedure 73 agarose gel. Store extracts at 4°C for short-term use or −20°C for long-term storage.
Expected outcome. 50 µL of resuspended DNA in TE buffer. Yield and fragment size vary by tissue and run roughly 50-70% of a matched CTAB extraction:
| Tissue | Typical yield (ng) | Typical N50 (kb) | Notes |
|---|---|---|---|
| Fish muscle | 5,000-25,000 | 10-25 | Cleanest substrate; the "if this fails, chemistry is broken" positive control |
| Dried mushroom | 500-5,000 | 8-15 | Rehydration helps; bead beating helps more |
| Plant seed | 500-2,000 | 5-15 | Hardest substrate; without LN₂, fragment length takes the biggest hit |
| Dried insect | 300-2,000 | 5-15 | Yield tracks tissue mass closely |
| Pollen pellet (from honey) | 30-300 | 3-10 | Smallest input; fragmentation cumulative with pollen recovery handling |
QC pipeline. After extraction, measure on procedure 84 (Qubit fluorometric dsDNA quant — to be submitted alongside this one) and run 3-5 µL on procedure 73 agarose gel alongside a 1 kb+ DNA ladder. A260/A230 and A260/A280 on a NanoDrop are useful for purity assessment if available.
Direct use. 1-2 µL per 25 µL PCR reaction for amplicon work. For ONT RBK114 library prep, check fragment size on gel first — if N50 < 5 kb, consider switching to procedure 82 (CTAB) output from the same tissue batch.
Storage. 4°C for 1 week, −20°C for 1 year+. Avoid freeze-thaw cycles for HMW applications; aliquot if planning multiple downstream uses.
Troubleshooting.
| Symptom | Likely cause | Fix |
|---|---|---|
| No pellet after isopropanol | Low DNA input; tissue not fully lysed | Extend lysis to 120 min; bead-beat tissue if not already; verify Proteinase K stock is active |
| Cloudy supernatant after salt-precip spin | Incomplete protein pelleting | Extend centrifuge to 15 min at 14,000 × g; chill supernatant on ice an additional 10 min and re-spin |
| Sticky gelatinous pellet | Polysaccharide carryover (common on seeds, mushrooms) | Second salt-precipitation step on the supernatant before isopropanol; additional 70% EtOH wash |
| Low A260/A230 (< 1.8) | Carbohydrate or carryover | Re-precipitate with 0.1 vol 3 M NaOAc pH 5.2 + 2.5 vol ethanol; extra 70% EtOH wash |
| Low A260/A280 (< 1.7) | Residual protein | Longer proteinase K step (add 5 µL fresh, 30 min at 55°C); 2nd salt-precip cycle |
| SDS precipitates in lysis tube | Buffer cooled below 15°C | Warm lysis buffer to 37°C before use; keep reactions above 25°C during setup |
| Smeared band on gel, low N50 | DNA shearing or incomplete lysis | Use wide-bore or cut pipette tips during all transfers; avoid vortex after lysis starts |
| Failed downstream PCR | Inhibitor carryover | Dilute template 1:10 before PCR; additional SPRI cleanup (procedure 74) |
| Clear improvement switching to procedure 82 | Tissue chemistry genuinely needs CTAB/chloroform | Note it in the POC comparison — this is exactly the data we're collecting |
References
- Miller SA, Dykes DD, Polesky HF (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research 16(3):1215. (Original salting-out method paper — the template for this protocol). DOI paper
- Aljanabi SM, Martinez I (1997). Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Research 25(22):4692-4693. (Cross-organism salt-extraction variant — validation across animal/plant/microbial substrates). DOI paper
- LibreBiotech procedure 82 — CTAB DNA Extraction (Cross-Tissue Reference Protocol) — the lab-grade chemistry paired against this one in the POC factorial. Link protocol
- LibreBiotech procedure 71 — Chelex-100 DNA Extraction (adjacent alternative for PCR-ready short-fragment extractions). Link protocol
- LibreBiotech procedure 73 — Agarose Gel Electrophoresis (fragment-size QC for extracts). Link protocol
- LibreBiotech procedure 72 — Standard PCR Setup and Thermocycling (downstream amplicon workflow consuming extracts). Link protocol