TITLE: HMW gDNA purification proto
AUTHORS: Ivette Cornejo Corona, Devon Boland, tpd

[DESCRIPTION]
Optimized protocol for efficient extraction of HMW gDNA from the polysaccharide-rich microalga Botryococcus, enabling long-read sequencing on the Oxford Nanopore Technologies platform. 

[BEFORE_START]
Ensure Bench Cleanliness: Prior to starting the protocol, thoroughly sanitize lab bench and instruments. 

[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. 

[STEPS]
SECTION: Biomass harvesting and HMW gDNA isolation
1. Biomass preparation 
• Harvest the biomass by filtration using a 10 μm nylon net.  
SECTION: Biomass pre-wash
2. • 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. 
SECTION: Extraction process
3. • 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. 
SECTION: Biomass harvesting and HMW gDNA isolation
1.2. • 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℃. 
SECTION: Biomass harvesting and HMW gDNA isolation
1.1. • 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. 
SECTION: RNA digestion
4. •  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. 
SECTION: HMW gDNA precipitation
5. • 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. 
SECTION: HMW gDNA wash and resuspension
6. • 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.