Oct 23, 2025
Version 1
DNA extraction from apple leaves or apple buds V.1
- 1Division of Science, New York Abu Dhabi – Saadiyat Island, Abu Dhabi – United Arab Emirates;
- 2Génétique Quantitative et Évolution – Le Moulon, Université Paris-Saclay, INRAE, CNRS, AgroParisTech, 91190 Gif-sur-Yvette –France
Protocol Citation: Anthony Venon, Sadaf Habib, Amandine Cornille 2025. DNA extraction from apple leaves or apple buds. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5koedv1b/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: October 17, 2025
Last Modified: October 23, 2025
Protocol Integer ID: 230077
Keywords: dna extraction from apple leaf, plant genomic dna extraction, dna extraction, dna purification step, cleaner dna, purified dna, elution of purified dna, nagel nucleospin plant ii kit, extraction, purity of dna, dna, apple leaf, qiagen tissuelyser ii, dna amount, efficient removal of contaminant, disrupted plant tissue, complex plant tissue, chloroform extraction, plant tissue, apple bud, input samples such as bud, downstream silica column
Abstract
This protocol describes a DNA purification step prior to following the standard workflow of the Macherey-Nagel NucleoSpin Plant II kit for plant genomic DNA extraction.
It is specifically designed for frozen and mechanically disrupted plant tissue, allowing efficient removal of contaminants such as polysaccharides, secondary metabolites, and cell wall debris that may interfere with downstream silica column binding.
The procedure begins with cryogenic grinding using a Qiagen TissueLyser II (or III), followed by chemical lysis using Carlson buffer and RNase A. A chloroform extraction is then performed to separate the aqueous phase from organic impurities. The supernatant is combined with PC buffer and applied to silica spin columns, followed by a series of ethanol-based wash steps and elution of purified DNA.
This enhanced pre-treatment improves the yield and purity of DNA from difficult or complex plant tissues and is fully compatible with the manufacturer’s protocol starting at the silica column binding step. It is also well suited for low-input samples such as buds, where DNA amounts are typically limiting. This approach consistently produces higher yields and cleaner DNA, enabling its use in downstream applications such as genotyping, PCR analysis, Illumina sequencing, or Oxford Nanopore Technologies (ONT) sequencing.
Guidelines
This protocol is intended for plant tissues that are frozen and require strong mechanical disruption for efficient DNA extraction.
Follow all biosafety and chemical safety procedures, particularly when handling chloroform.
For optimal yield, use pre-cooled metal adapters and maintain cryogenic conditions between homogenization steps.
This pre-treatment protocol leads into the Macherey-Nagel NucleoSpin‱ Plant II kit workflow at the silica binding step.
Materials
Materials and Reagents
Liquid nitrogen
Carlson lysis buffer
RNase A
PC buffer
PW1 buffer
PW2 buffer
PE (elution) buffer
Chloroform
1.5 mL microcentrifuge tubes
Green silica spin columns
Collection tubes (2 mL)
Filtered pipette tips
Ethanol (96–100%)
Gloves
Lab coat
Safety goggles
Equipment
TissueLyser II or III
Metal adapter plates for TissueLyser
Vortex mixer
Refrigerated centrifuge (11,000 × g)
Heating block or water bath (55–65 °C)
Chemical fume hood
Nanodrop spectrophotometer
Qubit fluorometer (optional)
Ice bucket
Cryogloves
Safety warnings
- Liquid nitrogen is extremely cold and can cause severe cryogenic burns. Always handle with cryogloves and face protection, and work in a well-ventilated area to avoid oxygen displacement.
- Chloroform is toxic, volatile, and potentially carcinogenic. Use only under a chemical fume hood, and wear appropriate PPE (gloves, lab coat, goggles).
- PC Buffer contains guanidinium chloride, a chaotropic and hazardous agent. Avoid skin contact and inhalation;
- Ensure that all ethanol is fully removed before elution. Residual ethanol can interfere with DNA quantification and downstream enzymatic reactions (e.g., PCR, digestion).
- Avoid excessive vortexing or forceful pipetting during lysis or elution to preserve high molecular weight DNA.
Ethics statement
This protocol involves the extraction of DNA from plant tissues only. No human or animal subjects are involved. Plant material was collected and processed in accordance with institutional and national guidelines.
Before start
- Pre-cool the metal adapters and tissue samples in liquid nitrogen.
- Preheat a water bath or heat block to 65 °C for lysis incubation.
- Preheat the elution buffer (PE) to 55 °C for the final elution step.
Prepare the following:
- Carlson lysis buffer
- Wash buffers PW2
Sampling
We collected apple buds and apple leaves in the field in 2mL tubes with stainless steel balls. The samples were then flash-frozen and stored at -80°C for long-term conservation.
Carlson Lysis Buffer - Day 0
Prepare 400 µL Carlson Lysis buffer per sample:
100 millimolar (mM) Tris-HCl 9.5 ,
2 Mass / % volume CTAB,
1.4 Molarity (M) NaCl
1 Mass / % volume PEG 8000,
20 millimolar (mM) EDTA.
Note
The buffer can be prepared in advance by batches of 1 or 2L.
Prepare the buffer at least one day in advance.
Lysis steps - Day 1
16m
Place the frozen samples into pre-cooled metal adapters compatible with the Qiagen TissueLyser II or III.
First homogenization round: Run the TissueLyser for 00:00:30 at 22 Hz.
30s
Immediately after the first run, immerse both the tube and the metal adapter in liquid nitrogen to re-freeze the sample and maintain cryogenic conditions.
⚠️ Note: If you are processing many samples or working slowly, it may be necessary to refresh the metal adapters in liquid nitrogen between runs to ensure optimal freezing.
Second homogenization round: Run the TissueLyser again for 00:00:30 at 22 Hz.
30s
After the second homogenization, immediately add 400 µL of Carlson lysis buffer to each sample tube.
Add 10 µL of RNase A (provided with the Macherey-Nagel NucleoSpin Plant II DNA extraction kit) to each tube.
Mix gently by pipetting up and down or briefly vortexing.
Incubate the samples at 65 °C for 00:15:00 to facilitate cell lysis and improve DNA yield.
15m
Purification steps
18m 15s
Working under a chemical fume hood, add 400 µL of chloroform to each lysed sample tube.
Vortex the samples vigorously for 00:00:15 to ensure complete mixing of the aqueous and organic phases.
15s
Centrifuge the tubes at 11000 x g, 4°C, 00:10:00 to separate the phases.
10m
While centrifuging, prepare the silica spin columns (green) by placing each one into a collection tube.
In a separate empty collection tube (without a column), add 450 µL of PC buffer (provided in the Macherey-Nagel kit).
After centrifugation, carefully collect the upper aqueous phase (avoid disturbing the interphase and organic layer).
Transfer the supernatant into the tube containing 450 µL of PC buffer.
Mix thoroughly by pipetting up and down several times.
Load 700 µL of the mixture onto the green silica spin column placed in its collection tube.
Centrifuge the silica column at 11000 x g, Room temperature, 00:01:00 .
1m
Discard the flow-through from the collection tube and place the column back into the same tube.
Add 400 µL of PW1 buffer to the column.
Centrifuge at 11000 x g, Room temperature, 00:01:00 .
1m
Discard the flow-through and return the column to the collection tube.
Add 650 µL of PW2 buffer to the column.
Centrifuge at 11000 x g, Room temperature, 00:01:00 .
1m
Discard the flow-through and return the column to the collection tube.
Add 200 µL of PW2 buffer again to the column.
Centrifuge at 11000 x g, Room temperature, 00:05:00 to ensure complete removal of residual ethanol.
5m
Discard the collection tube and transfer the column to a new, clean 1.5 mL microcentrifuge tube.
DNA Elution
Add 50 µL of pre-warmed elution buffer (PE buffer) directly to the center of the column membrane.
- Buffer should be preheated to 55 °C .
Incubate at room temperature for 5 minutes to allow elution.
Centrifuge at 11000 x g, Room temperature, 00:01:00 to collect the eluted DNA.
(Optional second elution): Pipette the eluate back onto the column membrane.
Wait an additional 5 minutes, then centrifuge again at 11000 x g, Room temperature, 00:01:00 .
Discard the column, and keep the eluate in the microcentrifuge tube.
DNA Quality and Quantity Assessment
Use a Nanodrop spectrophotometer and/or Qubit fluorometer to measure DNA concentration and assess purity.
Results
| Sample | Qubit (ng/µL) | Elution Volume (µL) | Quantity (µg) | Run_name | Flowcell_ID | P2_position | Barcode | |
| FR_E28_a | 160 | 50 | 8 | 251015_Applebuds_F_FRE28-E18-A4-ESC24 | PBG23388 | B | 13 | |
| FR_E18_b | 132 | 50 | 6.6 | 251015_Applebuds_F_FRE28-E18-A4-ESC24 | PBG23388 | B | 14 | |
| FR_A4_a | 83.4 | 50 | 4.17 | 251015_Applebuds_F_FRE28-E18-A4-ESC24 | PBG23388 | B | 15 | |
| ES_C24_b | 77.2 | 50 | 3.86 | 251015_Applebuds_F_FRE28-E18-A4-ESC24 | PBG23388 | B | 16 | |
| ES_E38_b | 67.8 | 50 | 3.39 | 251015_Applebuds_E_ESA23-C28-FRC12-ESE28 | PBG23439 | A | 11 | |
| ES_C28_b | 122 | 50 | 6.1 | 251015_Applebuds_E_ESA23-C28-FRC12-ESE28 | PBG23439 | A | 10 | |
| FR_C12_a | 139 | 50 | 6.95 | 251015_Applebuds_E_ESA23-C28-FRC12-ESE28 | PBG23439 | A | 12 | |
| ES_A23_b | 114 | 50 | 5.7 | 251015_Applebuds_E_ESA23-C28-FRC12-ESE28 | PBG23439 | A | 9 |
Table 1. DNA quantification and sample information for ONT sequencing.
Qubit-based quantification results for genomic DNA extracted from plant tissue samples using the enhanced pre-treatment protocol prior to the NucleoSpin Plant II workflow. Each sample is listed with its DNA concentration (ng/µL), elution volume (µL), total DNA yield (µg), and associated metadata including run name, flowcell ID, loading position (A/B), and barcode used for multiplexing.
Samples were multiplexed in groups of four using a ligation-based approach with the Oxford Nanopore SQK-NBD114.24 kit, and sequenced on a FLO-PRO114M flow cell using the P2-Solo device.
Figure 1. Summary of ONT sequencing run (position A, ES_A23_b; ES_C28_b; ES_E38_b; FR_C12_a samples)
Run performed on a FLO-PRO114M flow cell using the PromethION 2 Solo (P2-Solo) with 4 multiplexed samples (ES_A23_b; ES_C28_b; ES_E38_b; FR_C12_a). A total of 139.63 Gb of sequence data was generated over 72 hours, with 34.11 million reads. Basecalling success reached 100%, with 126.77 Gb of high-quality reads (Q ≥ 8). The read N50 was 12.13 kb.
Figure 2. Read length distribution and outlier analysis (position A, ES_A23_b; ES_C28_b; ES_E38_b; FR_C12_a samples)
The read length histogram for run shows a peak between 3–10 kb, with a long tail extending beyond 40 kb. The N50 of 12.13 kb confirms a robust proportion of long reads. Outlier analysis reveals several ultra-long reads exceeding 49 kb.
Figure 3. Summary of ONT sequencing run (position B, FR_E28_a; FR_E18_b; FR_A4_a; ES_C24_b samples)
Run performed on the same PromethION 2 Solo device using a second flow cell (FLO-PRO114M), loaded with 4 multiplexed (FR_E28_a; FR_E18_b; FR_A4_a; ES_C24_b). The run yielded 120.09 Gb of sequence data across 71 hours 59 minutes, totaling 28.7 million reads. Of those, 25.37 million reads (106.25 Gb) passed the Q8 quality threshold. The N50 was 11.88 kb, comparable to run A and suitable for downstream long-read analysis.