Total DNA isolation protocol

Practical extraction protocols suitable for diverse tissues (animal, plant, blood) and challenging samples.

Choose CTAB for broad compatibility; use the “difficult tissues” workflow for high polysaccharides/secondary metabolites, herbarium, and soil.

Scope and notes
  • Designed for small and large tissue samples (typical volume 100–200 μl).
  • Genomic DNA does not necessarily require “gentle mixing”; vortexing is acceptable for most workflows.
  • Use appropriate safety procedures for chloroform and chaotropic salts.

Contents

CTAB protocol for the isolation of DNA

Reagents

  • CTAB buffer: 2% CTAB, 1.5 M NaCl, 10 mM Na3EDTA, 0.1 M HEPES-acid
  • Chloroform:isoamyl alcohol (24:1)
  • 100% isopropanol
  • 70% ethanol
  • 1×TE (1 mM EDTA, 10 mM Tris-HCl, pH 8.0)
100 ml example: 2 g CTAB, 2.4 g HEPES-acid, 2 ml 0.5 M Na3EDTA, 30 ml 5 M NaCl.

Recommended equipment

  • 2 ml Safe-Lock tubes
  • 6 mm glass bead for grinding
  • Microcentrifuge (max speed)
  • 65°C incubator/heat block
  1. Grind tissue in a 2 ml tube with a 6 mm glass bead (e.g., mixer mill 2–10 min at 30 Hz).
  2. Add 1 ml CTAB buffer, mix (mixer mill 2 min at 30 Hz) and incubate at 65°C for 1 hour.
  3. Spin 2 min (max speed). Transfer clarified supernatant to a new 2 ml tube with an equal volume of chloroform.
  4. Mix thoroughly for 3 min (e.g., mixer mill at 30 Hz).
  5. Spin 2 min (max speed). Transfer the aqueous layer to a new tube containing 600 μl 2-propanol, mix well, then centrifuge 2 min (max speed).
  6. Discard supernatant. Wash pellet with 1.8 ml 70% ethanol, vortex well; centrifuge 2 min (max speed) and discard ethanol.
  7. Do not over-dry pellet. Dissolve immediately in 300 μl 1×TE (pH 8.0) at 55°C for 5–10 min.

CTAB protocol for difficult tissues (herbarium / soil)

Key idea

Use higher CTAB concentration and a silica spin column cleanup to handle polysaccharides and inhibitors.

Reagents

  • CTAB buffer: 3% CTAB, 1.5 M NaCl, 10 mM Na3EDTA, 0.1 M MOPS-acid
  • 96% ethanol
  • Buffer QG (guanidine thiocyanate-based)
  • Wash buffer PE
  • Elution buffer (Tris/EDTA, pH ~9.0)
100 ml example: 3 g CTAB, 2.1 g MOPS-acid, 2 ml 0.5 M Na3EDTA, 30 ml 5 M NaCl.

Columns (examples)

  • HiBind® DNA Mini column (Omega Bio-tek) or compatible
  • NucleoSpin® columns (MN) or compatible
  • Columns compatible with common Qiagen spin systems
  1. Grind herbarium sample (≤50 mg) with 6 mm bead (e.g., 15 min at 30 Hz).
  2. Add 1 ml CTAB buffer, vortex thoroughly; incubate at 65°C for 1–2+ hours.
  3. Spin 2 min (max speed). Transfer clarified supernatant to a new tube and add 96% ethanol (50–100% of CTAB volume). Mix.
  4. Load up to 700 μl onto a DNA mini column; spin 30 s. Repeat until all sample is loaded.
  5. Wash with 500 μl 96% ethanol; spin 30 s.
  6. Add 500 μl QG buffer; spin 30 s.
  7. Wash with 700 μl PE buffer; spin 30 s. Repeat wash once more, then spin 1 min to dry.
  8. Elute in a clean tube: add 200 μl elution buffer, heat 55°C for 10 min, spin 1 min. Store at −20°C.

Proteinase K method for DNA extraction

Extraction buffer (example)

0.8 M guanidine thiocyanate, 10 mM EDTA, 5% Tween 20, 0.5% Triton X-100, 50 mM HEPES-acid + Proteinase K (200 μg).
  1. Add 500 μl extraction buffer + Proteinase K; vortex thoroughly; incubate 37–55°C (overnight recommended) with occasional mixing.
  2. Add 700 μl chloroform; vortex 1 min to form an emulsion; optionally incubate 55°C for 30 min.
  3. Spin 5 min (max speed). Transfer supernatant to a new tube.
  4. Add 500 μl 2-propanol + 100 μl 3 M Na-acetate; mix; spin 4 min (max speed).
  5. Wash pellet with 1.8 ml 70% ethanol; spin 5 min; discard ethanol.
  6. Do not over-dry pellet; dissolve in 300 μl 1×TE (with RNase A if needed) at 55°C for 10–20 min.

Alternative Proteinase K buffers

  • 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0
  • 3 M guanidine hydrochloride, 10 mM EDTA, 5% Tween 20, 0.5% Triton X-100, 30 mM Tris-HCl, pH 8.0

References

Kalendar R. et al. (2021) Isolation and purification of DNA from complicated biological samples. Kalendar R. et al. (2023) Isolation of HMW DNA for long-read sequencing using gel electroelution trap. Kalendar R. et al. (2024) Improved nucleic acid isolation device using high-salt gel electroelution trap.

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