Feb 29, 2024
HMW gDNA purification proto
Peer-reviewed method
- Ivette Cornejo Corona1,
- Devon Boland1,
- Timothy Devarenne1
- 1Texas A&M University
External link: https://doi.org/10.1371/journal.pone.0301680
Protocol Citation: Ivette Cornejo Corona, Devon Boland, Timothy Devarenne 2024. HMW gDNA purification proto. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkoz2xv5r/v1
Manuscript citation:
Cornejo-Corona I, Boland DJ, Devarenne TP (2024) Method for isolation of high molecular weight genomic DNA from Botryococcus biomass. PLOS ONE 19(7). doi: 10.1371/journal.pone.0301680
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 29, 2024
Last Modified: February 29, 2024
Protocol Integer ID: 95973
Keywords: high molecular weight DNA, Botryococcus braunii, long-read sequencing
Abstract
Optimized protocol for efficient extraction of HMW gDNA from the polysaccharide-rich microalga Botryococcus, enabling long-read sequencing on the Oxford Nanopore Technologies platform.
Attachments
Guidelines
Maintain Frozen Samples: It is crucial to keep samples frozen during maceration using liquid nitrogen. Prepare sterile mortar, pestle, and spatulas for this step.
Handle Homogenization with Care: Exercise caution with the use of liquid nitrogen during maceration and sampling preparation.
Optimize Polysaccharide Removal: For effective polysaccharide removal with minimal DNA damage, keep the sonication step prior to cell lysis brief and at a low power setting.
Monitor Pellet Size: After each sorbitol wash step, check for an increase in pellet size.
Buffer Preparation: Prepare and sterilize buffers in advance to streamline the process.
Warm DNA Extraction Buffer: Warm the DNA extraction buffer to 65°C for at least 5 minutes before use for optimal results.
Materials
A. Buffer preparation
1. Sorbitol wash buffer. Autoclave and store this buffer at 4°C for no more than six months.
• 100 mM Tris-HCl pH 8.0
• 0.35 M Sorbitol
• 5 mM EDTA pH 8.0
• 1 % (W/V) Polyvinylpyrrolidone molecular weight 40,000 (PVP-40)
• 1% (V/V) 2-Mercaptoethanol (β-ME). Note: Add after autoclaving and before use. It is best to aliquot the amount of buffer needed and add β-ME to this aliquot.
2. DNA extraction buffer. Autoclave and store this buffer at room temperature for no more than six months.
• 100 mM Tris-HCl pH 8.0
• 3M NaCl
• 3% CTAB
• 20 mM EDTA
• 1% (W/V) Polyvinylpyrrolidone
• 1% (V/V) 2-Mercaptoethanol (β-ME). Note: Add after autoclaving and before use. It is best to aliquot the amount of buffer needed and add β-ME to this aliquot.
3. 24:1 CHCl3/IAA buffer. Store this buffer at 4°C.
• 96 ml Chloroform
• 4 ml Isoamyl Alcohol.
4. 3M Sodium acetate buffer
• 408.3 g sodium acetate
• 3H2O per L
• pH to 5.2
• autoclave
5. 1x TE (Tris EDTA) Buffer
• 1mM EDTA, pH 8.0
• 10 mM Tris-HCl, pH 8.0
B. Culturing Botryococcus culturing
• Culture Botryococcus species of choice in 1 L roux flasks with 750 ml modified Chu 13 medium, pH 7.5.
• Maintain at 22°C under continuous aeration with 2.5% CO2.
• Grow cultures for 6 weeks under a 12 h light:12 h dark cycle using 13 W compact fluorescent 65 K lighting at an intensity of 280 μmol photons/m2/s.
Before start
Ensure Bench Cleanliness: Prior to starting the protocol, thoroughly sanitize lab bench and instruments.
Biomass harvesting and HMW gDNA isolation
Biomass harvesting and HMW gDNA isolation
Biomass preparation
• Harvest the biomass by filtration using a 10 μm nylon net.
• Collect small amounts of biomass from the mesh using a rubber spatula and immediately freeze by placing in a 50 ml Falcon tube containing liquid nitrogen.
• Repeat until all biomass is collected into a single Falcom tube.
• Store at -80℃ until needed.
• Place small amount of frozen biomass into mortar and pestle with liquid nitrogen.
• Grind biomass until a fine powder is formed, keeping frozen at all times.
• Weigh out ~100 mg aliquots, pace in 1.5 ml eppendorf tube, and store at -80℃.
Biomass pre-wash
Biomass pre-wash
• Add 1 ml sorbitol wash buffer to 1.5 ml eppendorf tube containing ~100 mg ground biomass.
• Allow sample to thaw while vortexing for 10 seconds
• Keep samples on ice and sonicate for 25 seconds at 30% of power.
• Centrifuge at 2,500 x g for 5 minutes at room temperature. Discard the liquid phase by aspiration or decanting. Save the pelleted and floating biomass.
• Repeat the biomass pre-wash step three times.
Extraction process
Extraction process
• Add 700 μl DNA extraction buffer pre-warmed to 65°C, homogenize by vortexing for 10 seconds.
• Incubate at 65°C for 30 minutes mixing by inversion every 10 minutes.
• Incubate samples at room temperature for 5 minutes.
• Add 700 μl CHCl3:IAA buffer, vortex for 10 seconds, and centrifuge at 2,500 x g for 10 minutes at room temperature.
• Carefully transfer the upper aqueous phase (approximately 500 μl) to a new 1.5 ml eppendorf tube and keep on ice.
RNA digestion
RNA digestion
• Add 2 μl RNase A (25 mg/ml), and incubate at 37°C for 15 minutes mixing by inversion every 5 minutes.
• Add 500 μl CHCl3:IAA buffer, vortex 5 seconds, and centrifuge at 13,000 x g for 10 minutes at 4°C. • Transfer the upper phase to a new 1.5 ml eppendorf tube and keep on ice.
HMW gDNA precipitation
HMW gDNA precipitation
• Precipitate the HMW gDNA by adding 0.1 volumes of 3M sodium acetate pH 5.2 and 0.66 volumes of cold (-20°C) isopropanol.
• Incubate samples overnight at -20°C.
• Centrifuge at 13,000 x g for 10 minutes at 4°C, and discard supernatant by aspiration or decanting. • Dry pellet by resting inverted on paper towels at room temperature.
• Wash dried pellets with 1 ml of 70% ethanol and invert several times.
• Centrifuge at 13,000 x g for 10 min at 4°C. Remove the supernatant by aspiration to avoid pellet disturbance.
• Dry samples in a vacuum centrifuge for 10 min at 36°C.
• Resuspend HMW gDNA by adding 100 μl 1x TE buffer. Incubate at room temperature for 10 min then gently homogenize by inversion.
• Avoid pipetting that will shear the DNA.
• Store at -80°C until needed.