Apr 24, 2026

CTAB-Based Protocol for Genomic DNA Extraction from Fungal and Bacterial Samples

DOI
https://dx.doi.org/10.17504/protocols.io.ewov1rwrplr2/v1
  • Wellington Santos Fava1,
  • James Venturini1
  • 1School of Medicine, Federal University of Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul, Brazil
Wellington Santos Fava
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DOI: https://dx.doi.org/10.17504/protocols.io.ewov1rwrplr2/v1
Protocol Citation: Wellington Santos Fava, James Venturini 2026. CTAB-Based Protocol for Genomic DNA Extraction from Fungal and Bacterial Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1rwrplr2/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working.
Created: April 23, 2026
Last Modified: April 24, 2026
Protocol  Integer ID: 315621
Keywords: proteinase K, phenol–chloroform extraction, mechanical disruption, DNA precipitation, spectrophotometric analysis, genomic dna extraction, protocol for genomic dna extraction, standardized method for genomic dna extraction, extracted dna, organic purification, bacterial biomass, fungal, using ctab, bacterial samples this protocol, bacterial sample, chloroform extraction, quality dna, quantitative pcr, ctab
Abstract
This protocol presents a standardized method for genomic DNA extraction from fungal and bacterial biomass using CTAB-based lysis and organic purification. The procedure integrates mechanical disruption, proteinase K digestion, and phenol–chloroform extraction, followed by alcohol precipitation to obtain high-quality DNA. The extracted DNA is compatible with downstream applications including PCR, quantitative PCR (qPCR), electrophoresis, and other molecular analyses.
Guidelines
APPLICATION
Applicable to the processing of fungal and bacterial samples.
Materials
Materials
  • 2.0 mL tubes
  • 1.5 mL tubes
  • Filter tips
  • Sterile loop
  • Ice
Equipment
  • Micropipettes
  • Automated homogenizer
  • Dry bath (60 °C and 37 °C)
  • Refrigerated microcentrifuge
  • Vortex mixer
  • NanoDrop spectrophotometer
  • Electrophoresis system

CTAB extraction buffer

  • 2% CTAB
  • 1.4 M NaCl
  • 100 mM Tris-HCl, pH 8.0
  • 20 mM EDTA
Other reagents
  • Proteinase K
  • Phenol:chloroform:isoamyl alcohol (25:24:1)
  • 5 M ammonium acetate
  • Isopropanol
  • Cold 70% ethanol
  • Ultrapure water or TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA)
  • GelRed
Safety warnings
BIOSAFETY
  • Mandatory use of lab coat, gloves, and safety goggles.
  • Phenol/chloroform must be handled in a chemical fume hood.
  • Dispose of chemical waste according to institutional regulations.
  • Follow the applicable biosafety level
PRINCIPLE
The method is based on:

- Cell lysis with CTAB under high ionic strength
- Protein digestion with proteinase K
- Mechanical disruption using metal beads
- Purification with phenol:chloroform:isoamyl alcohol
- Alcohol precipitation of DNA
PROCEDURE
Sample preparation

Add to a 2 mL tube:
  • A loopful of fungal biomass
  • 490 μL CTAB buffer
  • 10 μL proteinase K
  • 6 steel beads
Homogenization Homogenize under the following conditions:
  • 3 cycles
  • 3400 rpm
  • 30 s each
  • 30 s intervals
Maximum time before the next step: 5 minutes.
Lysis Incubate at 60 °C for 60 min

Maximum time before the next step: 10 minutes.
Organic extraction Add 500 μL phenol:chloroform:isoamyl alcohol Mix briefly (vortex) Centrifuge:
  • 15 min
  • 12,000 rpm
  • 4 °C
Contaminant precipitation Transfer 500 μL of the supernatant to a new 1.5 mL tube Add 225 μL of 5 M ammonium acetate Mix by inversion Incubate on ice for 30 min 12. Centrifuge:
  • 10 min
  • 12,000 rpm
  • 4 °C
DNA precipitation Transfer 500 μL of the supernatant to a new 1.5 mL tube Add 275 μL isopropanol Mix by inversion Centrifuge:
  • 5 min
  • 12,000 rpm
  • 4 °C
Washing Discard the supernatant Add 500 μL cold 70% ethanol Centrifuge:
  • 5 min
  • 12,000 rpm
  • 4 °C
Remove the ethanol completely using a pipette tip after a quick spin.
Drying Dry at 37 °C for 10–15 min

Do not exceed this time.
Elution
Add 50 μL ultrapure water or TE buffer
Incubate at 37 °C for 20 min
DNA ANALYSIS
Electrophoresis

  • 3 μL DNA + 3 μL GelRed
  • 120 V for 50 min
Criterion: intact high-molecular-weight band.
Quantification (NanoDrop)

Acceptable ranges:
  • 260/280: 1.8–2.0
  • 260/230: ≥ 1.8 (ideal > 2.0)
Minimum concentration:
  • PCR: ≥ 10 ng/μL
  • qPCR: ≥ 20 ng/μL
STORAGE
DNA stored at −20 °C
CRITICAL POINTS
  • Excess biomass reduces purity.
  • Degraded phenol compromises DNA.
  • Residual ethanol inhibits PCR.
  • Over-dried pellet hinders elution.