Feb 06, 2026
Phytophthora capsici CTAB DNA Extraction for High-Molecular Weight DNA
- 1Universidad Nacional Autónoma de México
Protocol Citation: Pablo Vargas-Mejía 2026. Phytophthora capsici CTAB DNA Extraction for High-Molecular Weight DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl48rxjvo5/v1
Manuscript citation:
Modified from protocols.io (dx.doi.org/10.17504/protocols.io.3rsgm6e)
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: February 06, 2026
Last Modified: February 06, 2026
Protocol Integer ID: 242789
Keywords: phytophthora capsici ctab dna extraction for high, phytophthora capsici ctab dna extraction, phytophthora capsici, dna extraction, based dna extraction, molecular weight dna this protocol, mg of mycelium, mycelium, molecular weight dna, dna integrity, dna, ctab, majority of dna
Abstract
This protocol describes a CTAB-based DNA extraction optimized for Phytophthora capsici from as little as ~20 mg of mycelium, modified from protocols.io (dx.doi.org/10.17504/protocols.io.3rsgm6e), to maximize DNA integrity and purity while minimizing mechanical shearing and co-extraction of polysaccharides and phenolic contaminants. Typical yields are ~50 µL at ~300 ng/µL, with the majority of DNA >25 kb when handled without vortexing and with minimal pipetting.
Guidelines
Storage and handling notes:
If the CTAB buffer precipitates during storage, warm at 55–65 °C and mix until fully dissolved before use.
Use freshly prepared or properly stored antioxidants; add β-mercaptoethanol immediately before extraction.
Materials
- CTAB DNA Extraction Buffer (pre-warmed to 55 °C)
- Chloroform:Isoamyl alcohol (24:1)
- RNase A (20 mg/mL)
- 3 M Sodium acetate, pH 5.2
- Ethanol 100% (ice-cold)
- Ethanol 70% (ice-cold)
- TE buffer (recommended) or nuclease-free water
- Thermomixer (55 °C)
- Microcentrifuge capable of ~16,000 × g
- 1.5 mL microcentrifuge tubes; disposable pestles
- NaCl 8.3 g 1.4 M
- EDTA 0.744 g ~20 mM (depends on EDTA form/MW)
- CTAB 2 g 2% (w/v)
- PVP 2 g 2% (w/v); binds polyphenolics
- Ascorbic acid 0.088 g Antioxidant; typo fix from 0.88 g
- dH2O To 100 mL Bring to final volume
- β-mercaptoethanol 0.1–0.2% (v/v) Add fresh before use; handle in hood
CTAB DNA Extraction Buffer (100 mL final volume)
Prepare the buffer without β-mercaptoethanol, then add β-mercaptoethanol fresh immediately before use.
| A | B | C | |
| Component | Amount for 100 mL | Final concentration / note | |
| Tris-HCl pH 8.0 (1 M) | 10 mL | 100 mM | |
| NaCl | 8.3 g | 1.4 M | |
| EDTA | 0.744 g | ~20 mM | |
| CTAB | 2 g | 2% (w/v) | |
| PVP | 1 g | 2% (w/v); binds polyphenolics | |
| Ascorbic acid | 0.088 g | Antioxidant | |
| dH2O | To 100 mL | Bring to final volume | |
| β-mercaptoethanol | 0.1–0.2% (v/v) | Add fresh before use; handle in hood |
Safety warnings
- Avoid vortexing after lysis; mix by slow inversion or gentle flicking to prevent DNA shearing.
- Use wide-bore tips (or cut tips) when transferring viscous lysates; minimize pipetting up/down.
- Avoid disturbing the interphase during chloroform steps; carryover reduces purity and inhibits enzymes.
- Do not overdry the DNA pellet; overdried pellets can be difficult to dissolve and reduce recovery.
Before start
Prepare the buffer without β-mercaptoethanol, then add β-mercaptoethanol fresh immediately before use.
Procedure
8h 53m
Pre-warm CTAB buffer: Preheat CTAB DNA Extraction Buffer to 55 °C until fully dissolved.
10m
Homogenize mycelium (gentle): Grind Phytophthora mycelium (20-50 mg ) in a 1.5 mL tube preloaded with 200 µL warm CTAB buffer using a sterile pestle. Once homogeneous, add 500 µL more CTAB buffer (total 700 µL ) and mix gently by inversion or flicking (do not vortex).
5m
Lyse: Incubate 00:30:00 at 55 °C with gentle shaking (200 rpm ) in a thermomixer.
30m
First chloroform extraction: In a fume hood, add 500 µL of chloroform:isoamyl alcohol (24:1). Mix by inversion for 00:03:00 (do not vortex).
3m
Clarify: Centrifuge 00:10:00 at maximum speed (~16000 x g ).
10m
Recover aqueous phase: Transfer ONLY the upper aqueous phase to a new tube, avoiding the interphase.
2m
RNase treatment: Add 5 µL RNase A (20 mg/mL). Mix gently and incubate 00:15:00 at 37 °C . Optionally add 5 µL of Proteinase K (20 mg/mL).
15m
Second chloroform extraction: Add500 µL of chloroform:isoamyl alcohol (24:1). Mix by slow inversion 00:03:00 (no vortex).
3m
Clarify (2): Centrifuge 00:10:00 at maximum speed (~16000 x g ).
10m
Transfer again: Transfer the upper aqueous phase to a NEW tube, avoiding any interphase.
2m
Precipitate DNA (salt + ethanol): Add 0.1 volume of 3 M sodium acetate pH 5.2 (e.g., 50 µL per 500 µL aqueous). Mix by inversion. Then add 500-1000 µL of ice-cold 100% ethanol and invert gently until mixed.
2m
Cold incubation: Precipitate at 80 °C for 30-00:45:00 (or at 20 °C for 60 min).
45m
Pellet DNA: Centrifuge 00:15:00 at maximum speed. Note pellet location by orienting tube hinges outward.
15m
Wash pellet (70% ethanol): Remove supernatant carefully. Add 500 µL ice-cold 70% ethanol, invert once or twice, and centrifuge 00:05:00 at maximum speed.
5m
Repeat wash: Repeat the 70% ethanol wash once more.
1m
Dry: Remove all ethanol with a fine tip. Air-dry 10–00:15:00 until ethanol odor is gone; do not overdry.
15m
Resuspend: Resuspend DNA in 50 µL TE buffer (37 °C recommended) or nuclease-free water. Dissolve at 4 °C for several hours or overnight with occasional gentle tapping.
6h
Quantify and QC: Use NanoDrop primarily for purity ratios (A260/280, A260/230). Run a 0.4% agarose gel overnight.
Protocol references
Modified from CTAB DNA Extraction for high quality/molecular weight DNA (dx.doi.org/10.17504/protocols.io.3rsgm6e)