canonical: https://aviantestpro.com/knowledge/quality-control/dna-extraction-sop
1. Scope
This SOP describes the protocol for extracting genomic DNA from avian feather follicle samples for downstream PCR-based applications including CHD sexing, pathogen detection, microsatellite genotyping, and performance gene SNP analysis. The protocol is suitable for feathers from psittacines, pigeons, passerines, galliformes, anseriformes, and other avian species. It is intended for use by trained laboratory personnel.
2. Principle
Feather follicle cells are lysed using proteinase K digestion in a chaotropic buffer containing guanidine hydrochloride. The lysis buffer denatures proteins and inactivates nucleases, protecting released DNA. DNA is selectively bound to a silica membrane in the presence of high salt concentrations, washed with ethanol-based buffers to remove proteins, polysaccharides, and other contaminants, and eluted in a low-ionic-strength buffer (low-salt Tris-EDTA). The purified DNA is suitable for PCR amplification, restriction digestion, and sequencing applications.
3. Materials & Equipment
| Item | Specification | Supplier | Storage |
|---|
| Proteinase K | > 600 mAU/mL, recombinant (DNase/RNase-free) | SENO Reagent | −20°C; stable 6 months |
| Lysis Buffer | 4 M guanidine HCl, 25 mM EDTA, 1% Triton X-100, 50 mM Tris pH 7.5 | SENO Reagent | Room temp |
| Wash Buffer 1 | 5 M guanidine HCl, 30% ethanol, 10 mM Tris pH 7.5 | SENO Reagent | Room temp |
| Wash Buffer 2 | 80% ethanol, 10 mM Tris pH 7.5 | SENO Reagent | Room temp (store sealed to prevent evaporation) |
| Elution Buffer | 10 mM Tris-HCl, 0.5 mM EDTA, pH 8.5 | SENO Reagent | Room temp |
| Binding Buffer | 6 M guanidine HCl, 50% isopropanol | SENO Reagent | Room temp |
| Silica Column | 2 mL collection tube assembly | SENO Reagent | Room temp |
| Heating Block | 56°C ± 1°C | Lab equipment | Calibrated quarterly |
| Microcentrifuge | ≥ 12,000 × g, rotor for 1.5/2.0 mL tubes | Lab equipment | Calibrated annually |
| Pipettes | 10, 100, 200, 1000 µL, calibrated | Lab equipment | Calibrated quarterly |
| Vortex Mixer | — | Lab equipment | — |
| Scalpel / Scissors | Sterile, disposable | Lab consumable | Single use |
| Filtered Pipette Tips | DNase/RNase-free | Lab consumable | — |
| 1.5 mL Microcentrifuge Tubes | DNase/RNase-free | Lab consumable | — |
4. Protocol
- Equilibrate heating block to 56°C (± 1°C) — verify with calibrated thermometer
- Prepare Proteinase K working solution: reconstitute at 20 mg/mL in 50 mM Tris pH 8.0, 1 mM CaCl₂; aliquot and store at −20°C
- Label all tubes with sample ID, date, and technician initials — use barcode labels if LIMS-integrated
- Wipe work surface with 10% bleach followed by 70% ethanol
- Inspect feather samples: confirm 3–5 intact follicles visible (white bulb-like base)
- If sample is a single small feather (canary/finch), proceed with 1 feather; note in extraction log
4.2 Tissue Lysis
| Step | Action | Duration | Notes |
|---|
| 4.2.1 | Cut follicle tips (2–3 mm section containing bulb) into 1.5 mL microcentrifuge tube | — | Use sterile scalpel; change blade between samples |
| 4.2.2 | Add 200 µL Lysis Buffer | — | Dispense against tube wall; do not touch pipette tip to follicle |
| 4.2.3 | Add 20 µL Proteinase K working solution | — | Mix by pulse-vortex 5 s at medium speed |
| 4.2.4 | Incubate at 56°C in heating block | 30–60 min | Vortex every 15 min until tissue fully digested; no visible fragments remain |
| 4.2.5 | Add 200 µL Binding Buffer | — | Mix by inversion 10× or pulse-vortex 5 s |
| 4.2.6 | (Optional) Centrifuge at 12,000 × g for 2 min to pellet debris | 2 min | Only if sample contains melanin pigment (visible black particles) |
4.3 DNA Binding
| Step | Action | Duration | Notes |
|---|
| 4.3.1 | Transfer lysate to silica column assembly (column + 2 mL collection tube) | — | Avoid touching membrane with pipette tip |
| 4.3.2 | Centrifuge at 10,000 × g | 1 min | Room temperature |
| 4.3.3 | Discard flow-through | — | Reuse collection tube |
4.4 Washing
| Step | Buffer | Volume | Centrifuge | Notes |
|---|
| 4.4.1 | Wash Buffer 1 | 500 µL | 10,000 × g, 1 min | Discard flow-through after spin |
| 4.4.2 | Wash Buffer 2 | 500 µL | 10,000 × g, 1 min | Discard flow-through after spin |
| 4.4.3 | Wash Buffer 2 | 500 µL | 10,000 × g, 1 min | Repeat wash — removes residual guanidine |
| 4.4.4 | Empty spin (dry column) | — | 12,000 × g, 2 min | Removes residual ethanol; critical for downstream PCR |
| 4.4.5 | Open column lid and air-dry | 2–3 min | Room temp | Ensures complete ethanol evaporation |
4.5 Elution
| Step | Action | Duration | Notes |
|---|
| 4.5.1 | Transfer column to clean, labeled 1.5 mL elution tube | — | Label tube before transfer |
| 4.5.2 | Add 50 µL Elution Buffer directly to membrane center | — | Pre-warm to 56°C for 15–30% higher yield |
| 4.5.3 | Incubate at room temperature | 5 min | Do not exceed 10 min |
| 4.5.4 | Centrifuge at 12,000 × g | 2 min | Collect eluate |
| 4.5.5 | Re-apply eluate to membrane, repeat incubation and spin | — | Increases yield ~15% (optional, recommended for low-yield samples) |
| 4.5.6 | Label elution tube: sample ID, date, DNA ID, technician initials | — | Barcode if available |
5. Quality Assessment
| Parameter | Method | Acceptance Criteria | Action if Failed |
|---|
| Concentration | NanoDrop A₂₆₀ | ≥ 10 ng/µL (preferred); ≥ 5 ng/µL (minimum) | Re-extract with increased follicle count (5–7 feathers) |
| Purity — protein | A₂₆₀/A₂₈₀ | 1.7–2.0 | If < 1.7: repeat wash steps; if > 2.0: RNA contamination (treat with RNase) |
| Purity — chaotropic salt | A₂₆₀/A₂₃₀ | ≥ 1.5 | If < 1.5: guanidine carryover; re-purify with additional wash or ethanol precipitation |
| Integrity | Agarose gel (0.8%, visualize with GelRed) | High MW band > 10 kb (no smearing below 500 bp) | Degraded sample; discard and request re-collection |
| PCR amplifiability | CHD PCR (P2/P8 primers) | Amplification in ≥ 95% of samples from a batch | Check extraction SOP compliance; retrain if batch failure > 5% |
Quality Assessment Workflow
| Decision Point | Action |
|---|
| All parameters pass | Store at 4°C (short-term ≤ 1 month) or −20°C (long-term); proceed to PCR |
| Concentration < 5 ng/µL | Re-extract with larger follicle sample; if repeat fails, request fresh sample |
| A₂₆₀/A₂₈₀ < 1.6 | Repeat wash protocol on existing column eluate; re-assess |
| A₂₆₀/A₂₃₀ < 1.2 | Ethanol precipitation or re-purify on fresh column |
| Degraded (no high-MW band) | Discard sample; request fresh collection with documented storage conditions |
6. Troubleshooting
| Problem | Likely Cause | Solution | Cross-Reference |
|---|
| Low yield (< 5 ng/µL) | Insufficient follicle material | Use 5–7 feathers instead of 3–5 | PCR Troubleshooting § 1.1 |
| Low yield | Old / improperly stored feathers | Request fresh sample; verify collection date < 30 days | Sample Guide |
| Low yield | Incomplete lysis | Increase proteinase K incubation to 60 min; vortex more frequently | — |
| No DNA detected | Very old sample (> 6 months) | Request fresh sample; sample type may degrade after 60 days at room temp | — |
| A₂₆₀/A₂₈₀ < 1.6 | Protein contamination | Add 5 µL extra proteinase K, double incubation time | PCR Troubleshooting § 1.2 |
| A₂₆₀/A₂₃₀ < 1.5 | Guanidine carryover | Repeat wash with Wash Buffer 2 + dry spin 3 min | — |
| Column clogged | Excess tissue | Reduce follicle amount (2–3 mm is sufficient); pre-digest larger follicles with extra PK | — |
| A₂₆₀/A₂₈₀ > 2.0 | RNA contamination | Add 1 µL RNase A (10 mg/mL), incubate 10 min at 37°C, then re-purify | — |
| PCR fails despite good QC | Inhibitor not detected by spectrophotometry | Run 1:10 dilution of DNA; if amplifies → inhibition confirmed | PCR Troubleshooting § 2.2 |
7. Expected Yield
| Sample Type | Typical Yield (ng/µL in 50 µL) | Total Yield (µg) | Quality Notes |
|---|
| Parrot feather (3–5 follicles) | 15–50 | 0.75–2.5 | Generally high quality |
| Pigeon feather (3–5 follicles) | 10–40 | 0.5–2.0 | Good for PCR + genotyping |
| Canary/finch feather (5–7 follicles) | 5–20 | 0.25–1.0 | Small follicles; use more feathers |
| Duck/goose feather (1–2 follicles) | 30–80 | 1.5–4.0 | Large follicles; excellent yield |
| Blood (FTA card, 1 punch, 3 mm) | 20–60 | 1.0–3.0 | Consistent; preferred for genotyping |
| Blood (liquid EDTA, 50 µL) | 30–100 | 1.5–5.0 | Highest yield; must ship cold |
8. Waste Disposal
| Waste Type | Disposal Method |
|---|
| Used silica columns | Incinerate (biohazard waste) |
| Lysate + flow-through | Decontaminate with 10% bleach (1 hour contact), then drain |
| Scalpel blades | Sharps container |
| Gloves + pipette tips | Biohazard bag → incineration |
9. Document History
| Version | Date | Author | Changes |
|---|
| 1.0 | 2025-01 | SENO QC | Initial release |
| 1.1 | 2025-06 | SENO R&D | Modified binding buffer composition for increased yield; added re-apply elution step |
| 1.2 | 2025-12 | SENO R&D | Added FTA card processing instructions; updated expected yield table |
Cross-References
This SOP is a controlled document. Unauthorized modification is prohibited. For protocol revisions, contact [email protected].