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CTAB DNA Extraction (Cross-Tissue Reference Protocol)

High-molecular-weight genomic DNA extraction using cetyltrimethylammonium bromide (CTAB) lysis, chloroform phase separation, and isopropanol precipitation. Single unified protocol parameterised for five starting materials — honey (pollen pellet from procedure 84), dried insects, plant seeds, dried mushrooms, and fish tissue. Produces DNA suitable for fluorometric quantification, fragment-size QC on an agarose gel, and downstream ONT long-read sequencing. Reference lab-grade chemistry for the Stage 0.5 extraction-feasibility POC. Written for a novice audience.

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Version History

Version 0.1.2 Latest locked

Effective: 2026-04-28

Tagged 10 process-scoped extraction-condition parameters with scope='process' to leverage new spec-layer scope distinction (LibreBiotech commits a485a63 + 3cd09de). Sample-scoped params (tissue_type, tissue_mass_mg) unchanged. Bridge change only; protocol body identical to v0.1.1.

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Version 0.1.1 Viewing locked

Effective: 2026-04-21

Dev review 2026-04-21 folded in: (1) chloroform default flipped to chloroform:IAA 24:1 v/v for HMW preservation (IAA suppresses foaming-induced shearing); neat chloroform moved to substitution note. (2) Rotator mixer (equipment_types id 7) added for gentle phase-separation mixing. (3) Forward-reference IDs corrected: pollen recovery 83→84, Qubit 84→85. (4) 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

Effective: 2026-04-21

Initial release. Reference lab-grade extraction chemistry for the Stage 0.5 Cross-Tissue Extraction Feasibility POC. Unified CTAB protocol parameterised across five tissue types; tissue-specific preparation captured in the tissue_type parameter and the preparation notes. Doyle & Doyle 1987 method (~39 years old, field-standard).

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Procedure Details

Safety & Hazards

BSL-1 work only. Food-grade or similarly unhazardous biological starting materials. Not validated for clinical, regulated, or infectious samples.
Chloroform. Hepatotoxic, suspected carcinogen (IARC Group 2B), volatile. Handle only in a fume hood. Use nitrile gloves (latex is permeable). No open bench work.
β-mercaptoethanol (β-ME). Acutely toxic if inhaled/ingested; extreme stench at trace levels. Handle in a fume hood. 0.2% working dilution is less hazardous than the neat reagent but still requires ventilation. DTT can substitute (0.1% final) if β-ME access is restricted — chemistry is equivalent for most substrates.
Liquid nitrogen. Cryogen (−196°C). Splash-proof goggles, cryo-gloves, no sealed containers. Ventilation adequate — LN₂ displaces O₂ and causes asphyxiation in enclosed spaces.
Isopropanol and ethanol. Flammable. No open flame near precipitation or wash steps. Solid ignition sources (hot plates, Bunsen burners) need to be absent from the precipitation step.
CTAB powder. Cationic detergent, skin/eye irritant as dry solid. Weigh in a fume hood or with a dust mask; buffer stock solutions are benign.
Pipetting hygiene. Filter tips throughout to prevent aerosol carryover between samples; critical for the POC's cross-tissue comparison.
Tissue handling. Grinding fresh tissue in liquid N₂ generates aerosols — keep a lid on the mortar once the LN₂ has boiled off.
Not a clinical extraction. The output is research-grade genomic DNA; not validated for diagnostic or forensic use.

Preparation Notes

CTAB extraction buffer (make fresh weekly, store at room temperature):

2% CTAB (w/v)
100 mM Tris-HCl pH 8.0
20 mM EDTA
1.4 M NaCl
Sterilise by autoclaving (121°C, 20 min). Do not add β-ME or DTT until immediately before use — reducing agents oxidise on storage and lose potency.

Adjust pH with HCl/NaOH before autoclaving using a pH meter. Mix thoroughly on a magnetic stirrer with gentle heat (50°C) — CTAB takes 15-20 min to fully dissolve at 2% w/v.

Working lysis buffer (per-batch): 600 µL of CTAB buffer per sample + 0.2% (v/v) β-mercaptoethanol added immediately before use. Pre-warm to 65°C.

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

Chloroform:isoamyl alcohol (24:1, v/v): the lab-grade default. IAA at 24:1 dilution suppresses foaming during phase separation; foaming is a known HMW-shearing and yield-loss mechanism in long-read extraction workflows. Typically purchased as a pre-mixed stock (Sigma C0549 or equivalent); alternatively, mix 24 parts chloroform + 1 part isoamyl alcohol (3-methyl-1-butanol) in-house. Substitution note: neat chloroform works if IAA is unavailable, but accept higher risk of foaming-induced HMW shearing — flag for the troubleshooting table and use gentle inversion rather than vortex to compensate.

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

Tissue	Mass input	Preparation	Notes
Pollen pellet (from honey, procedure 84)	Full pellet	Resuspend pellet in 100 µL CTAB buffer before continuing	No grinding needed; pollen walls are rigid but the heat step opens them
Dried insects	10-50 mg whole body or ~1 leg of a large specimen	Crush in microtube with a clean plastic pestle, or bead-beat 30 s at 5,000 RPM	Chitin fragments at lysis; don't need complete homogenisation
Plant seeds	50-100 mg	Flash-freeze in liquid N₂, grind to fine powder in a pre-chilled mortar and pestle	Hard coat + polysaccharides: the LN₂ grind is load-bearing
Dried mushrooms	50 mg	Cut into small pieces with a clean blade; optional rehydration in buffer 5 min	Fungal chitin softens quickly once hydrated
Fish tissue (fresh, frozen, or ethanol-preserved)	20-50 mg	Mince with a clean blade	Soft tissue; no LN₂ needed

Mental model. CTAB binds DNA reversibly in high-salt (1.4 M NaCl) conditions, separating it from polysaccharides and polyphenols that are the main quality enemies of plant/fungal DNA prep. The reducing agent (β-ME or DTT) keeps tissue proteins denatured and breaks disulfide bonds; Proteinase K digests remaining protein; chloroform phase-separates everything lipid-like and protein-like into the organic layer; DNA stays in the aqueous phase and precipitates in isopropanol. The 70% ethanol wash desalts the pellet without redissolving the DNA.

What CTAB gets you that Chelex doesn't. Procedure 71 Chelex produces fast, PCR-ready template with short fragments (typically < 5 kb). CTAB produces high-molecular-weight DNA (> 20 kb, often > 40 kb) suitable for long-read sequencing. Use CTAB when the downstream needs HMW input (ONT library prep, archival genomic DNA); use Chelex when speed and PCR-readiness are the priorities.

Timing

Tissue preparation: 10-20 min per sample depending on tissue (pollen pellets are fastest; seed grinding is slowest).
Lysis: 60 min at 65°C (longer — up to 2 h — for recalcitrant tissues).
Phase separation + precipitation: 30-40 min active.
Washing + drying + resuspension: 20 min.
Total per batch: ~2.5-3 h active for 8 samples in parallel.
Heat blocks support 12-sample batches. Centrifuge slot count is usually the bottleneck; 6-slot microcentrifuges process ~6 samples in parallel at each spin step.

Equipment (Catalog) 14

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
Alternative to mortar/pestle for insect / mushroom tissues
Mortar and pestle
Specs: Ceramic, chilled in advance for LN₂ grinding
Seeds only; pre-cool in -80°C freezer for 30 min or by LN₂ bath before use
Benchtop centrifuge Centrifugation
Specs: 15 mL / 50 mL conical tube capable (optional)
For scaling honey dissolution / pollen recovery upstream
Microcentrifuge Centrifugation
Specs: ≥12,000 × g; room-temperature capable
Main workhorse
Micropipette Liquid handling
Specs: P20, P200, P1000; filter tips throughout
Wide-bore tips for aqueous phase transfer; cut tips for final resuspension
pH meter Measurement
Specs: Benchtop or pocket; calibrate with pH 4/7/10 buffers
CTAB buffer pH adjustment
Spectrophotometer Measurement
Specs: UV-capable (A260/A230/A280); optional
Nanodrop-style for post-extraction purity QC
Magnetic stirrer Mixing
Specs: With gentle heat (50°C) capable
CTAB buffer preparation — CTAB takes 15-20 min to dissolve at 2% w/v
Rotator mixer Mixing
Specs: Low speed (5-10 RPM) capable
Gentle rotation during the 2-3 min chloroform-phase-separation mix — preserves HMW DNA vs. manual inversion for batches
Vortex mixer Mixing
Specs: Standard benchtop
Used for buffer mixing only; NOT for DNA during or after lysis
Fume hood Safety
Specs: Ducted or filtered; adequate face velocity
Required for chloroform + β-ME handling
Heat block Thermal regulation
Specs: 65°C + 37°C capable
Primary lysis at 65°C; resuspension option at 37°C
Water bath Thermal regulation
Specs: 65°C capable (alternative to heat block)
For batches > 12 samples where heat block slots are full

Materials (Catalog) 14

Chloroform Chemical
Qty: 600 µL
Chloroform:isoamyl alcohol (24:1, v/v) is the lab-grade default for HMW preservation (pre-mixed Sigma C0549 or equivalent). Neat chloroform is an acceptable substitute if IAA is unavailable — note higher risk of foaming-induced HMW shearing; compensate with gentle inversion only.
CTAB Chemical
Qty: 12 mg
2% w/v in lysis buffer; 600 µL buffer per sample
EDTA Chemical
Qty: 12 µmol
20 mM in lysis buffer
Ethanol Chemical
Qty: 1000 µL
70% v/v, freshly diluted; 500 µL per wash × 1-2 washes
Isopropanol Chemical
Qty: 350 µL
0.7 volumes of recovered aqueous phase; room temperature
Liquid nitrogen Chemical
Qty: ~50 mL (seeds only)
Seed grinding only; ~50 mL per mortar/pestle session
NaCl Chemical
Qty: 840 µmol
1.4 M in lysis buffer
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
β-mercaptoethanol Chemical
Qty: 1.2 µL (0.2% v/v)
Add fresh immediately before use; DTT at 0.1% is an acceptable substitute
Filter pipette tips Consumable
Qty: 5 tips
Filtered tips for aqueous phase transfer + DNA handling to prevent aerosol carryover
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: 10 tips
Generic tips; ~10 per sample across all pipetting steps
Proteinase K Enzyme
Qty: 3 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 working CTAB lysis buffer (with β-ME or DTT added fresh) added per sample, in µL. 600 µL standard; scale with tissue mass for large inputs.
`lysis_temp_c`	number	—	`65`	degree Celsius (UO:0000027)	Lysis incubation temperature in °C. 65°C is optimal for CTAB + Proteinase K activity.
`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, dried mushrooms).
`reducing_agent`	text	—	`BME`	—	Reducing agent in the working lysis buffer. Valid values: BME (β-mercaptoethanol, 0.2% v/v), DTT (dithiothreitol, 0.1% w/v). Chemically equivalent for most substrates.
`proteinase_k_final_mg_ml`	number	—	`0.1`	—	Final Proteinase K concentration in the lysis tube, in mg/mL. 0.1 mg/mL standard; up to 0.5 mg/mL for heavy-protein tissues.
`chloroform_volume_ul`	number	—	`600`	—	Volume of chloroform:isoamyl alcohol (24:1, v/v) added for phase separation, in µL. Equal volume to lysis buffer (600 µL) standard. Neat chloroform is an acceptable substitute at the same volume if IAA is unavailable.
`isopropanol_volume_ul`	number	—	`350`	—	Volume of isopropanol for precipitation, in µL. 0.7 × recovered aqueous phase (~500 µL) = 350 µL standard.
`ethanol_wash_cycles`	number	—	`1`	—	Number of 70% ethanol wash cycles. 1 standard for clean tissues (fish); 2 for high-polyphenol 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.
`pre_grind_with_ln2`	boolean	—	`false`	—	Whether to flash-freeze and grind tissue in liquid nitrogen before lysis. true for plant seeds; false for all other tissues.

Procedure Steps (Version 0.1.1)

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. For seed samples, grind in a pre-chilled mortar and pestle under liquid nitrogen to a fine powder and transfer immediately to the tube before thawing.

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 CTAB lysis buffer to 65°C in a water bath or heat block. Prepare the working lysis buffer by adding β-mercaptoethanol to 0.2% v/v (or DTT to 0.1% w/v) immediately before use — reducing agents oxidise in stored buffer.

Add 600 µL of pre-warmed working lysis buffer to each sample tube. Mix gently by inverting 5× or tapping; do not vortex yet (HMW DNA shears under mechanical agitation).

Add 3 µL of 20 mg/mL Proteinase K stock (final concentration ~0.1 mg/mL). Mix gently by inversion.

Incubate at 65°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, dried mushrooms), extend to 90-120 minutes.

Cool tubes to room temperature (2-5 minutes on bench). Transfer to a fume hood for the chloroform step. Add 600 µL of chloroform:isoamyl alcohol (24:1, v/v) to each tube — equal volume to lysis buffer. Cap firmly. (Neat chloroform is an acceptable substitute if IAA is unavailable — see preparation notes.)

Mix by gentle inversion for 2-3 minutes — thorough mixing is essential for phase separation, but vortexing shears HMW DNA. A tube rotator at low speed works well for batches.

Centrifuge at 12,000 × g for 10 minutes at room temperature. The aqueous (upper) phase contains the DNA; the organic (lower) phase and white interface contain proteins, lipids, and polysaccharides.

Carefully transfer ~500 µL of the upper aqueous phase to a new labelled 1.5 mL microcentrifuge tube using a wide-bore pipette tip or a cut P1000 tip. Avoid disturbing the white interface — any transfer of interface material brings polysaccharides into the DNA prep.

Add 0.7 volumes (typically 350 µL) of room-temperature isopropanol. Mix by gentle inversion for 30 seconds. Incubate at room temperature for 5 minutes — DNA pellets do not improve with longer incubation at this stage.

Centrifuge at 12,000 × g for 10 minutes at room temperature. A white or translucent pellet should form at the bottom or side of the tube. Remove supernatant by pipetting; do not pour off (pellets can be loose).

Wash the pellet by adding 500 µL of freshly-prepared 70% ethanol (do not mix or vortex — the wash is a surface desalting, not a redissolve). Centrifuge at 12,000 × g for 5 minutes. Remove supernatant carefully.

Repeat the 70% ethanol wash once more for samples with visible carryover or high-polyphenol tissues. Otherwise proceed to drying.

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 can be 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 HMW DNA. Pipetting up and down will shear the DNA; use gentle 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, buffer lot) as process_actuals. Output sample material_type should be extract.

Quantify DNA via procedure 85 (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.

Completion Notes

Expected outcome. 50 µL of resuspended DNA in TE buffer. Yield and fragment size vary substantially by tissue:

Tissue	Typical yield (ng)	Typical N50 (kb)	Notes
Fish muscle	10,000-50,000	> 40	Cleanest substrate; positive control
Dried mushroom	1,000-10,000	20-40	Good if LN₂-ground or heat-lysed fully
Plant seed	1,000-5,000	10-30	Hardest substrate; A260/A230 often < 2.0
Dried insect	500-3,000	10-25	Yield limited by tissue mass
Pollen pellet (from honey)	50-500	5-20	Smallest input; fragment size often modest

QC pipeline. After extraction, measure on procedure 85 (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 to assess fragment size. 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; 200-400 ng per barcode for ONT RBK114 library prep.

Storage. 4°C for 1 week, −20°C for 1 year+. Avoid repeated freeze-thaw cycles for HMW applications — aliquot if planning multiple downstream runs.

Troubleshooting.

Symptom	Likely cause	Fix
No pellet after isopropanol	Low DNA input; tissue not fully lysed	Extend lysis to 90-120 min; increase tissue mass; verify lysis buffer was pre-warmed
Brown/green pellet	Polyphenol / pigment carryover (plant/fungal tissue)	Add 2% PVP-40 to lysis buffer; repeat chloroform step; additional 70% EtOH wash
Sticky gelatinous pellet	Polysaccharide carryover (common on seeds, mushrooms)	2nd chloroform phase separation before precipitation; high-salt precipitation (2.5 M NaCl, not isopropanol)
Low A260/A230 (< 1.8)	Carbohydrate or phenol contamination	Re-precipitate with 0.1 vol 3 M NaOAc pH 5.2 + 2.5 vol ethanol; longer 70% EtOH wash
Low A260/A280 (< 1.7)	Residual protein	Longer proteinase K step (add 5 µL fresh, 30 min at 65°C); repeat chloroform
Smeared band on gel, low N50	DNA shearing during handling	Cut pipette tips wide-bore; invert rather than vortex during lysis; avoid aggressive pipetting
Failed downstream PCR	Inhibitor carryover	Dilute template 1:10 or 1:100 before PCR; additional SPRI cleanup (procedure 74)
Failed ONT library prep	Fragment size too short	Verify HMW band on gel before committing library reagents. If using neat-chloroform substitution, foaming-induced shearing is the likely cause — switch to default chloroform:IAA 24:1 and use gentle inversion only (no vortex) during phase separation.

References

Doyle JJ, Doyle JL (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19:11-15. (Original CTAB method paper; the procedural template for this protocol). paper
Porebski S, Bailey LG, Baum BR (1997). Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter 15:8-15. (Reference for PVP and salt-precipitation adjustments in the troubleshooting table). DOI paper
Chen H, Rangasamy M, Tan SY, Wang H, Siegfried BD (2010). Evaluation of five methods for total DNA extraction from western corn rootworm beetles. PLoS ONE 5:e11963. (Reference for CTAB applicability to insect tissue). DOI paper
LibreBiotech procedure 71 — Chelex-100 DNA Extraction for PCR-Ready Template (adjacent alternative for fast PCR-ready extractions; shorter fragments). Link protocol
LibreBiotech procedure 73 — Agarose Gel Electrophoresis (fragment-size QC for CTAB extracts). Link protocol
LibreBiotech procedure 72 — Standard PCR Setup and Thermocycling (downstream amplicon workflow consuming CTAB extracts). Link protocol