Libre Biotech
Dashboard Projects Labbook Protocols Training Groups Sign in

High-Salt CTAB DNA Extraction

Obtain high-molecular-weight genomic DNA suitable for long-read sequencing or Southern blotting. The protocol combines a **2 % CTAB / 1.4 M NaCl / 1 % PVP** buffer (captures polysaccharides & polyphenols) with a **high-salt ethanol-sodium-citrate precipitation** that removes residual CTAB.

sample_prep
Version History
Version 1 Current
Effective: 2025-07-15

First version.

Procedure Steps (Version 1)

Reagents – preparation notes

Reagent Final composition Prep & storage
CTAB buffer 50 mM Tris-HCl pH 8.0, 20 mM EDTA, 1.4 M NaCl, 2 % CTAB, 1 % PVP-40 Heat to 60 °C to dissolve CTAB; add PVP last; store 4 °C ≤ 1 month
Lysis buffer 50 mM Tris-HCl pH 8.0, 20 mM EDTA, 200 mM NaCl, 2 % SDS, Proteinase K 20 mg mL⁻¹ Add Proteinase K right before use; store buffer 4 °C
RNase A 10 mg mL⁻¹ Boil 15 min to inactivate DNase; aliquot, −20 °C
100 % Ethanol + 0.1 M sodium citrate 0.1 M Na₃C₆H₅O₇ in absolute ethanol Dissolve citrate in 10 mL water, bring to 1 L ethanol
70 % ethanol (fresh) – Mix 700 mL ethanol + 300 mL water

Equipment checklist

  • 1.5 mL nuclease-free tubes
  • RNase-free micropestle or LN₂ mortar & pestle
  • Bench-top centrifuge ≥ 14 000 ×g, 4 °C rotor
  • 65 °C dry block or water bath
  • −20 °C freezer space for overnight precipitation
  • Pipettes 20 µL – 1 000 µL + aerosol tips

Pre-setup (10 min)

  1. Preheat CTAB buffer to 65 °C (invert until clear).
  2. Chill 100 % ethanol/sodium-citrate to −20 °C.
  3. Label two sets of tubes per sample: “CTAB-1” and “DNA-final”.
  4. Wipe bench & tools with 70 % ethanol + RNase-away.

Homogenise tissue (5 min)

  1. Transfer 30–50 mg frozen insect tissue to a chilled 1.5 mL tube.

  2. Add 1.0 mL Lysis buffer.

  3. Grind with micropestle (30 s bursts) until no visible chunks. Solution turns cloudy white; that is SDS + proteins.

  4. Spin 5 min, 14 000 ×g, room temp; discard supernatant (removes lipids & pigments).

Tip: do NOT skip this SDS pre-wash—yields improve by 20 %.

CTAB extraction (30 min – 4 h)

  1. Add 1.0 mL pre-warmed CTAB buffer to pellet.
  2. Invert 10×; pellet should disperse.
  3. Add 2 µL RNase A (20 µg).
  4. Incubate 65 °C: Minimum 30 min (small fragments) – up to 4 h (HMW DNA). Swirl every 30 min; suspension becomes viscous (DNA in solution).

Phenol:Chloroform clean-up × 2 (15 min)

  1. Cool tube 2 min; add 1.0 mL phenol:chloroform (1 : 1).
  2. Invert gently 20× (no vortex → avoids shearing).
  3. Spin 5 min, 14 000 ×g, RT.
  4. Transfer upper aqueous layer (~800 µL) to fresh tube.
  5. Repeat extraction once more with equal volume phenol:chloroform.

What you should see: organic phase turns brownish; aqueous phase clear-yellow.

High-salt ethanol precipitation (overnight)

  1. To the pooled aqueous phase (~750 µL) add 2 volumes (1.5 mL) cold 100 % ethanol + 0.1 M sodium citrate.
  2. Invert 10×; DNA forms long white strings.
  3. Incubate −20 °C ≥ 2 h (optimum: overnight).

Recover DNA pellet

  1. Spin 15 min, 14 000 ×g, 4 °C. White pellet sticks to tube wall.
  2. Carefully decant supernatant; do not dislodge pellet.
  3. Add 1 mL 70 % ethanol; flick to wash; spin 5 min.
  4. Repeat 70 % wash once.
  5. Air-dry pellet 5 min (surface dull, but not cracked).

Warning: over-dry → pellet glass-hard, dissolves poorly.

Resuspend DNA (5 min)

  • Add 30–50 µL LC-MS-grade water.
  • Tap tube every 2 min; incubate 37 °C 5 min if pellet stubborn.
  • Store −20 °C (≤ 6 months) or 4 °C (≤ 1 week).

Quality control

Assay Expected
NanoDrop A₂₆₀/A₂₈₀ 1.8 ± 0.1
NanoDrop A₂₆₀/A₂₃₀ ≥ 2.0
Agarose 0.7 % Single band > 20 kb
Qubit dsDNA BR Yield 5–15 µg (50 mg larva)

Low 260/230 means phenol/CTAB carry-over → do extra phenol:chloro extraction.

Troubleshooting

Problem Cause Solution
Gel smear < 10 kb Mechanical shearing Skip vortex; pipette wide-bore tips
Pellet won’t dissolve Over-dry; residual salts Incubate 37 °C; add 2 µL 10 mM Tris pH 8
Brown pellet Polyphenols Check PVP stock; add 0.2 % β-mercaptoethanol to CTAB buffer
Emulsion after phenol step Syringe shake too vigorous Swirl gently; spin extra 2 min

Timing summary

Step Hands-on Passive
Grind + SDS wash 5 min 5 min spin
CTAB incubation – 0.5–4 h
Phenol cleans 10 min 10 min spins
Ethanol precipitate 5 min Overnight
Wash + dissolve 10 min 5 min dry
Total active 30–35 min + passive ≈ 9–14 h

Bench-side checklist

  • CTAB buffer clear at 65 °C, PVP fully dissolved.
  • Phenol:chloro stored dark, equilibrated pH 8.
  • Ethanol + sodium-citrate pre-chilled −20 °C.
  • No vortex after phenol addition—gentle inversion only.
  • Pellet dries ≤ 5 min.
  • Record yield, purity ratios & date in logbook.

With these detailed instructions—reagent prep, visual cues, and “why-it-matters” notes—a novice can reproducibly isolate high-quality genomic DNA from H. armigera using the high-salt CTAB method.