May 07, 2026

Genomic DNA extraction from Wahlenbergia (bluebell) leaves V.1

 Forked from CTAB genomic DNA extraction from Arabidopsis leaf material
DOI
https://dx.doi.org/10.17504/protocols.io.yxmvm1z36v3p/v1
  • 1The Australian National University
  • EBL_ANU
Diep R R Ganguly
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvm1z36v3p/v1
Protocol Citation: Diep R Ganguly, Simarpreet Gill 2026. Genomic DNA extraction from Wahlenbergia (bluebell) leaves. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm1z36v3p/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: In development
We are still developing and optimizing this protocol
Created: February 05, 2026
Last Modified: May 07, 2026
Protocol  Integer ID: 242660
Keywords: genomic DNA, ctab, Plant Leaf Tissue, Wahlenbergia, genomic dna extraction from wahlenbergia, wahlenbergia leaf tissue, wahlenbergia leaf tissue for use, genomic dna extraction, extraction of genomic dna, wahlenbergia, genomic dna, read sequencing, based extraction
Funders Acknowledgements:
Australian Research Council
Grant ID: DE240100184
Abstract
CTAB-based extraction of genomic DNA from Wahlenbergia leaf tissue for use in short- and long-read sequencing.
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Guidelines
Ensure all workbenches are clean and nuclease-free (e.g. stepwise wiping with 0.01% SDS and 70% ethanol). Buffers should be prepared carefully and be nuclease-free and sterile (e.g. autoclave or filter-sterilized).
Safety warnings
Work with 2-mercaptoethanol and chloroform in the fume hood. Wear gloves and safety glasses.
Before start
Ensure that your leaf tissue has been ground into a fine powder and is kept frozen in liquid nitrogen until you are ready to add CTAB lysis buffer.
Cell lysis
15m
Prepare lysis buffer.
AB
Reagent[Cf]
cetyl trimethylammonium bromide (CTAB)2% (w/v)
NaCl2 M
Tris-Cl (pH 8)100 mM
EDTA (pH 8)20 mM
PVP-401% (w/v)
Lysis buffer recipe

15m
Aliquot the required volume of lysis buffer (550 µL per sample) and add the following:
  • 2-mercaptoethanol (0.5% v/v)
  • RNase A (100-200 µg/mL)
  • Spermidine or spermine (50 µM, optional)
10m
Warm lysis buffer (60 ºC) and mix by gentle inversion to re-dissolve any precipitates.
Ensure that your plant material has been ground into a fine powder via mortal and pestle or with a TissueLyser (do not let material thaw during this process).
Add 500-1,000 µL of warmed lysis buffer to frozen and ground tissue (500 µL per 50 mg tissue).
2m
Mix vigorously for 10-15 seconds and ensure that all plant material is in solution.
5m
Incubate at 60 ºC for 1-3 hours with periodic mixing every ~20-30 minutes (or use a benchtop tube rotator or ThermoMixer if available).
1h
Allow samples to cool to room temperature.
10m
Phase separation
15m
Add an equal volume of chloroform:IAA (24:1) and mix thoroughly for 10-15 seconds.
5m
Incubate for 3 minutes.
Centrifuge samples for 10 minutes at 20,000 rcf.
10m
Transfer the upper aqueous phase to clean tube (~400 µL).
5m
Repeat steps 8-11 on the recovered aqueous phase (be more conservative, e.g. ~300 µL).
20m
Precipitation
15m
Add 0.1-volume of 3 M Na0Ac (pH 5.2).
2m
Add 1-volume of 2-propanol (or 3-volumes of ethanol to avoid excess salts if sample volumes permit).
Mix by gentle inversion.
Incubate for 30-60 mins at room temperature (1-12 h at -20 °C if using ethanol).
1h
Centrifuge for 20 min at 20,000 rcf.
15m
Discard supernatant being careful to not disturb the pellet.
2m
Resuspension
17m
Add 750 µL of 70% ethanol and mix by inversion.
2m
Centrifuge samples at 9,200 rcf for 10 mins.
5m
Remove as much ethanol as possible by pipetting, then air dry pellet for 2-3 minutes.
10m
Resuspend DNA pellet in nuclease-free water or Tris (10 mM Tris-Cl, pH 8).
Incubate at 50 °C to help dissolve DNA if needed.
Determine yield, purity, and size distribution of samples using a Nanodrop and gel/capillary electrophoresis.
Protocol references
Inglis PW, Pappas M de CR, Resende LV, and Grattapaglia D (2018) Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications. PLOS ONE, 13, e0206085.
Jones A, Torkel C, Stanley D, Nasim J, Borevitz J, Schwessinger B (2021) High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. PLOS ONE 16(7): e0253830.
Acknowledgements
We acknowledge helpful suggestions from Ashley Jones.