Dec 17, 2025
Version 1
High Molecular Weight DNA Extraction from Cannabis sativa for Long-Read Sequencing V.1
- Brett Pike1,
- Claudia Cuervo2,
- Wilson Terán1
- 1Plant Biology and Productive Systems Group, Department of Biology, Pontifical Xavierian University, Bogotá, Colombia;
- 2Department of Microbiology, Pontifical Xavierian University, Bogotá, Colombia
Protocol Citation: Brett Pike, Claudia Cuervo, Wilson Terán 2025. High Molecular Weight DNA Extraction from Cannabis sativa for Long-Read Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6ek95gqe/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: December 16, 2025
Last Modified: December 17, 2025
Protocol Integer ID: 235148
Keywords: high molecular weight dna extraction from cannabis sativa, genomic dna from cannabis sativa plant tissue, high molecular weight dna extraction, dna extraction, cannabis sativa plant tissue, genomic dna, cannabis sativa, read sequencing, isolating high molecular weight, dna, nuclease contamination, sequencing platform, sequencing, high molecular weight, nuclei isolation
Abstract
This protocol describes a method for isolating high molecular weight (HMW) genomic DNA from Cannabis sativa plant tissue suitable for long-read sequencing platforms. The protocol uses nuclei isolation to minimize nuclease contamination and mechanical homogenization with the Precellys Evolution system. Expected yield: ~4 μg HMW DNA per gram of young shoot tissue.
Guidelines
- Nuclei isolation is critical to remove nucleases from vacuoles
- Protocol optimized for long-read sequencing of highly duplicated genes (e.g., cannabinoid synthases)
- Work performed under Resolution 1164 of 2021, Ministry of Justice, Republic of Colombia
Materials
**Equipment**
- Precellys Evolution homogenizer (Bertin Technologies)
- Microcentrifuge (refrigerated, capable of 12,000g)
- Centrifuge (capable of 1,500g, refrigerated to 4°C)
- Heat block or water bath (65°C)
- Vortex mixer
- Liquid nitrogen container
**Consumables**
- For 1g tissue: 6× reinforced 2mL tubes (P000943-LYSK0-A) OR 1× 7mL tube (P000944-LYSK0-A)
- 2.8mm stainless steel beads (P000925-LYSK0-A)
- 60 μm and 30 μm cell strainers/filters
- Disposable pipettes and wide-bore pipette tips
- 1.5-2 mL microcentrifuge tubes
- Plastic inoculation loop or p200 tip
**Reagents**
- Nuclei Isolation Buffer (NIB)**
- Prepare according to Circulomics Nuclei Isolation Protocol
- Store at 4°C
- Homogenization Buffer (HB)**
- Prepare 1L of 1× solution (as per Edwards 1991)
- Contains PVP (polyvinylpyrrolidone) and BME (β-mercaptoethanol)
- Store at 4°C
- Edward's Lysis Buffer**
- 200 mM Tris-HCl pH 7.5
- 250 mM NaCl
- 25 mM EDTA
- 0.5% SDS
- Other Reagents**
- Proteinase K
- 1M KCl
- RNase (DNase-free)
- Chloroform (CHCl₃)
- Saturated NaCl solution
- 95% ethanol and 70% ethanol
- EB buffer (10 mM Tris-HCl pH 8.0, no EDTA)
- Liquid nitrogen
Troubleshooting
Safety warnings
⚠️ Chloroform**: Use in fume hood. Toxic and potentially carcinogenic.
⚠️ Liquid nitrogen**: Wear cryogenic gloves and face protection. Can cause severe cold burns.
⚠️ β-mercaptoethanol (BME)**: Toxic. Use in fume hood with appropriate PPE.
PART 1: Nuclei Isolation
Select young shoot tips (preferred tissue type)
_Optional: Incubate plants in darkness for 3 days prior to extraction to reduce carbohydrate content_
Divide 1g fresh tissue among six 2mL tubes with 2.8mm metal beads, OR use one 7mL tube with 2.8mm metal beads
Snap-freeze tissue in liquid nitrogen
Homogenize frozen samples using Precellys Evolution at 6,200 RPM for 5 seconds
_Tip: Keep samples frozen until homogenization to minimize nuclease activity._
Add 1.6 mL Nuclei Isolation Buffer (NIB) to each tube and resuspend thoroughly
_Note: Buffer will likely freeze on contact with frozen sample_
Incubate with gentle shaking at room temperature for 15 minutes
Filter sample sequentially through 60 μm filter, then 30 μm filter into new 2 mL tubes
Pellet nuclei by centrifugation at 1,500g for 1 minute at 4°C. Remove supernatant.
Resuspend pellet in 1 mL ice-cold NIB by pipetting
Repeat steps 4-5 until supernatant is absolutely clear (typically 2-5 washes)
Resuspend pellet in 1 mL ice-cold Homogenization Buffer (HB) and centrifuge at 1,500g for 1 minute at 4°C
Remove HB and either proceed to DNA purification or store nuclei pellet at -80°C
_Checkpoint: Nuclei pellet should be free of green pigmentation and supernatant should be clear_
PART 2: DNA Purification
Resuspend nuclei pellet in 30 μL Proteinase K and vortex strongly for 20 seconds
_Critical: Ensure thorough resuspension so each nucleus is surrounded by proteinase_
Add 400 μL Edward's buffer and vortex briefly to mix
_Important: Beyond this point DNA is fragile. Use wide-bore tips and handle gently._
Incubate at 65°C for 30 minutes to 2 hours
Pellet cellular debris at 12,000g for 5 minutes
Transfer 400 μL supernatant to new tube, avoiding pellet
Add 200 μL of 1M KCl, mix by gentle inversion, and incubate at room temperature for 5 minutes
Centrifuge at 12,000g for 5 minutes and transfer 500 μL supernatant to new tube
Add 2.5 μL RNase, mix by inversion, and incubate at room temperature for 5 minutes
Add 500 μL chloroform and incubate at room temperature for 10 minutes with occasional inversion (perform in fume hood)
Separate phases at 5,000g for 1 minute
Transfer 400 μL aqueous (upper) layer to new tube. Do not disturb precipitate at interphase.
Add 200 μL saturated NaCl and mix by gentle inversion
Add 600 μL 95% ethanol and mix by inversion. DNA gel should become visible.
Hook out DNA using plastic inoculation loop or p200 tip and transfer to tube with 1 mL 70% ethanol
Incubate at room temperature for 10 minutes
Hook DNA into another tube with 1 mL fresh 70% ethanol
Centrifuge at 1,000g for 1 minute
Remove ethanol and air dry DNA gel at room temperature for 30 minutes (gel should appear clear)
Resuspend in 100 μL EB buffer (10 mM Tris-HCl pH 8.0, no EDTA)
Incubate at room temperature overnight to allow DNA to relax before quality control
_Final Checkpoint: DNA should be in solution and appear viscous_
Expected Results
Yield: ~4 μg HMW DNA per 1g young Cannabis sativa shoots
Quality Metrics:
- 260/280 ratio: ~1.8 (indicating pure DNA)
- 260/230 ratio: e2.0 (indicating removal of contaminants)
DNA Integrity: Should show high molecular weight fragments ( 3e50 kb) suitable for Oxford Nanopore and PacBio sequencing
Protocol references
33. Circulomics Nuclei Isolation Protocol (Circulomics, Baltimore, MD, USA)
34. Edwards K, Johnstone C, Thompson C. (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research 19(6):1349
Acknowledgements
Protocol developed at Pontifical Xavierian University and medcann pharma, Bogotá, Colombia. Equipment provided by Bertin Technologies (Precellys Evolution homogenizer).