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Chelex-100 DNA Extraction for PCR-Ready Template

Fast, low-cost DNA extraction suitable for PCR-based species identification and similar downstream assays. Uses Chelex-100 chelating resin + Proteinase K + heat lysis to yield PCR-ready template from small tissue samples (~20 mg) in under an hour. No toxic solvents, no spin columns, no purification steps — extract is used directly in PCR. Atomic technique for procedure 56 (COI Fish Barcoding) and any invertebrate / vertebrate tissue DNA workflow. Written for a novice audience.

sample_prep

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Effective: 2026-04-20

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Initial release. Atomic technique supporting the Sushi Truth pilot (procedure 56 COI) and general PCR-template preparation. Walsh et al. 1991 method (~34 years old, field-standard). Fresh original prose.

Procedure Details

Safety & Hazards

BSL-1 work only. Standard food-grade or similarly-unhazardous biological materials. Not validated for clinical, regulated, or infectious samples.
95–100°C heat step. Use a dedicated heat block with closed lids; use tube tongs; eye protection.
Proteinase K. Protease enzyme; handle with gloves to prevent skin degradation.
Chelex-100. Cation-chelating resin. Non-toxic but eye/skin irritant as dry powder. Prepare 5% w/v suspensions in a fume hood.
Pipetting hygiene. Gloves + filter tips throughout; segregate pre-amp and post-amp workflows.
Not a clinical extraction. Chelex extracts contain PCR inhibitors suitable only for direct-template use; not validated for quantitative PCR or archival storage.

Preparation Notes

Chelex suspension prep. 5% w/v Chelex-100 in low-EDTA TE (10 mM Tris-HCl pH 8.0 + 0.1 mM EDTA). Keep continually mixed on a magnetic stirrer — resin settles within seconds and uneven aliquots give wildly variable extraction yields. Store at 4°C for months.

Proteinase K stock. 20 mg/mL in water, aliquot at −20°C, single-use only (repeated freeze-thaw reduces activity).

Sample specification:

~20 mg tissue (rice-grain size) per sample.
Fresh, frozen, or ethanol-preserved all work. Formalin-preserved samples DO NOT work — formalin cross-links DNA and blocks PCR.
Highly cooked or heavily processed samples yield low-quality DNA — use mini-barcode primers downstream if that's your input.

Mental model: Chelex absorbs divalent metal ions (Mg²⁺, Fe²⁺) that otherwise damage DNA during the heat lysis step. The result is DNA in solution, contaminated by salts and cell debris, but suitable for direct PCR where tolerant enzymes like Taq can amplify despite the background.

Timing

Per sample: 45 min active time (tissue prep + extraction steps).
Batch efficient: 12+ samples in parallel with a heat block — same hands-on time.
Time-sensitive: extract used within 4 hours ideally (fresh), or stored at −20°C for up to 1 year.

Equipment (Catalog) 4

Microcentrifuge Centrifugation
Specs: ≥12,000 × g
Standard benchtop
Micropipette Liquid handling
Specs: P200, P1000; filter tips throughout
Dedicated pre-PCR area
Vortex mixer Mixing
Specs: Standard benchtop
Essential — Chelex settles fast
Heat block Thermal regulation
Specs: 56°C and 95°C capable in same unit, or two blocks
Dry heat block preferred over water bath

Materials (Catalog) 2

Proteinase K Enzyme
Qty: 5 µL of 20 mg/mL stock
NEB P8107S or similar; single-use aliquots at −20°C
Lysis solution Reagent
Qty: 200 µL of 5% suspension
Bio-Rad 142-1253 or similar; 5% w/v in low-EDTA TE

Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns

Name	Type	Required	Default	Unit	Description
`chelex_concentration_pct`	number	—	`5`	—	Chelex-100 w/v concentration in suspension. 5% standard; higher percentages add noise without benefit.
`proteinase_k_concentration_mg_ml`	number	—	`0.5`	—	Final Proteinase K concentration in the extraction tube. 0.5 mg/mL typical; up to 1 mg/mL for tough or cooked samples.
`lysis_temp_c`	number	—	`56`	degree Celsius (UO:0000027)	Temperature for initial Proteinase K digestion. 56°C optimal.
`lysis_time_min`	number	—	`30`	minute (UO:0000031)	Duration of Proteinase K digestion. 30 min standard; 60–120 min for cooked or heavily processed samples.
`denature_temp_c`	number	—	`95`	degree Celsius (UO:0000027)	Temperature for Proteinase K inactivation + DNA release. 95°C standard; 100°C if heat block goes there.
`denature_time_min`	number	—	`10`	minute (UO:0000031)	Duration of 95°C step. 10 min standard; shorter (6 min) reduces DNA damage in delicate samples.
`tissue_amount_mg`	number	—	`20`	—	Tissue mass input. 20 mg standard (rice-grain size); more yields more DNA but more inhibitor carryover.

Procedure Steps (Version 0.1.0)

Collect 2–3 tissue pieces from the sample (~20 mg total, rice-grain size). Transfer to a labelled 1.5 mL microcentrifuge tube. Cut into smaller fragments with a clean blade if samples are large or fibrous.

Record sample metadata in LibreBiotech: claimed species (if applicable), vendor, collection date/location, price, photo, and any other sample-chain-of-custody information as Sample annotations.

Vortex the 5% Chelex-100 suspension thoroughly for 30 seconds immediately before pipetting — the resin settles fast, so every aliquot must come from a well-mixed slurry.

Add 200 µL of freshly-vortexed 5% Chelex-100 suspension to each sample tube.

Add 5 µL of 20 mg/mL Proteinase K stock (final concentration ~0.5 mg/mL).

Incubate at 56°C for 30 minutes in a heat block. Vortex briefly (2–3 seconds) once at the 15-minute mark to keep the Chelex suspended and the tissue agitated.

Transfer the tube to a 95°C heat block and incubate for 10 minutes. This step denatures Proteinase K and releases DNA by disrupting remaining cellular structures.

Remove from the heat block. Vortex for 10 seconds to ensure complete mixing.

Centrifuge at 12,000 × g for 2 minutes at room temperature. The Chelex resin and cell debris pellet at the bottom; clear supernatant contains the DNA.

Carefully transfer ~100 µL of the clear upper phase to a new labelled 1.5 mL tube. Avoid the Chelex pellet at the bottom — disturbance brings inhibitors into the extract. Tilt the source tube slightly and pipette from the top.

Record the extraction as a Process in LibreBiotech, linking to the Sample records for each input tissue and recording the tube-label / lot number of the Chelex and Proteinase K stocks used. This creates the ISA-canonical provenance for every downstream PCR.

Use fresh extract within 4 hours for best results, or store at −20°C for medium-term use (up to 1 year).

Completion Notes

Expected outcome. ~100 µL of clear supernatant containing PCR-ready DNA. Concentration typically 5–50 ng/µL (varies 10-fold depending on tissue quality). No Nanodrop measurement expected — Chelex extracts read low/noisy due to salt and debris.

Direct use. 1 µL of extract per 25 µL PCR reaction is a standard starting point. If PCR fails: dilute extract 1:10 in water (reduces inhibitor concentration), retry.

Storage. 4°C for 24 hours; −20°C for up to 1 year. Some long-term precipitation is expected — vortex briefly before use after freezing.

Troubleshooting.

Symptom	Likely cause	Fix
No PCR product	Insufficient DNA; Chelex interference	Dilute extract 1:10 and retry; verify tissue amount
PCR works for some samples not others	Inhibitor carryover from tissue	Dilute extract further (1:50); shorter Proteinase K step
Smeary PCR bands	Genomic DNA degradation	Shorter incubation at 95°C (6 min instead of 10); use larger tissue amount
No band in cooked samples	DNA too degraded for full-length amplicon	Switch to mini-barcode primers (targets 100–200 bp fragments)
Inhibition in sensitive downstream assays	Chelex carryover in supernatant	Careful transfer avoiding pellet; 10 µL less aliquot

References

Walsh PS, Metzger DA, Higuchi R (1991). Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10(4):506–13. (Original method paper). paper
LibreBiotech procedure 56 — COI DNA Barcoding for Consumer Fish Samples (downstream use case). Link protocol