TITLE: High Molecular Weight DNA Extraction From Leaf Tissue
AUTHORS: Patricia Lu-Irving, Susan Rutherford

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A protocol designed for isolation and purification of high molecular weight genomic DNA from ~1g of vascular plant leaf tissue, for linked and/or long-read sequencing. Used successfully with </span><span style = "font-style:italic;">Syzygium oleosum</span><span> (Myrtaceae), </span><span style = "font-style:italic;">Rhodamnia argentea</span><span> (Myrtaceae), </span><span style = "font-style:italic;">Telopea speciosissima</span><span> (Proteaceae). Sorbitol pre-wash together with higher salt and CTAB concentrations (per Thermo Fisher Application Note 52645, adapted from Inglis et al 2018 doi: 10.1371/journal.pone.0206085), followed by SPRI bead-based purification (adapted from Schalamun et al. 2018 doi: 10.1111/1755-0998.12938) help to ensure sufficient purity for most genomic sequencing applications.</span></div></div>

[STEPS]
?. [SPRI bead cleanup]
Preheat 10mM Tris-HCl, pH 8 to 50°C. Make fresh 70% EtOH.
?. [SPRI bead cleanup]
Add 1x volume of washed beads to 1x volume of DNA.
?. [SPRI bead cleanup]
Incubate 10 minutes at room temperature, gently inverting the tubes periodically.
?. [SPRI bead cleanup]
Touch spin in desktop centrifuge to draw bead suspension to bottom of tube.
?. [SPRI bead cleanup]
Place tube on magnet rack and wait until bead pellet forms on side of tube.
?. [SPRI bead cleanup]
Remove and discard supernatant.
?. [SPRI bead cleanup]
Add 1mL fresh 70% EtOH and slowly rotate the tubes 360° in the magnet rack to wash bead pellet.
?. [SPRI bead cleanup]
Remove and discard supernatant.
?. [SPRI bead cleanup]
Repeat EtOH wash (steps 6-7) once.
?. [SPRI bead cleanup]
Touch spin in desktop centrifuge to draw residual EtOH to bottom of tube, and remove and discard residual EtOH by pipetting, taking care to avoid disturbing the bead pellet.
?. [SPRI bead cleanup]
Allow beads to dry briefly (less than 1 minute), then add 50-100uL (depending on expected/desired DNA concentration) 10mM Tris-HCl, pH 8 (preheated to 50°C).
?. [SPRI bead cleanup]
Incubate at least 10 minutes at room temperature to elute. If elution is difficult due to high concentration of high molecular weight DNA (thick, fluffy bead pellet), incubate overnight at 37°C.
?. [SPRI bead cleanup]
Place tube on magnet rack and wait until bead pellet forms on side of tube.
?. [SPRI bead cleanup]
Transfer supernatant to new tube.
?. [SPRI bead cleanup]
Evaluate DNA quantity (yield) using Qubit and adjust concentration as necessary for sample submission guidelines. Evaluate DNA purity and integrity via OD260/280, 260/230 (protein, phenol, carbohydrate contamination) and agarose gel (qualitative size, RNA contamination).
?. [Extraction]
Evaluate DNA quantity (yield) using Qubit and evaluate DNA purity and integrity via OD260/280, 260/230 (protein, phenol, carbohydrate contamination) and agarose gel (qualitative size, RNA contamination).
?. [Extraction]
Re-suspend pellet in 100 µL 10mM Tris-HCl pH 8.
?. [Extraction]
Discard supernatant, and allow pellet to air-dry on bench (take care not to over-dry).
?. [Extraction]
Centrifuge tubes at 14,000 x g for 20 minutes.
?. [Extraction]
Discard supernatant. Add 1.5 mL of 77 % ethanol. Gently dislodge pellet by inverting/tapping, and leave in fridge for 20 minutes.
?. [Extraction]
Centrifuge tubes at 14,000 x g for 20 min.
?. [Extraction]
Discard supernatant. Add 1.5 mL ice cold 77 % ethanol, gently dislodge pellet by inverting/tapping, and leave in fridge overnight.
?. [Extraction]
Centrifuge tubes at 14,000 x g for 20 min.
?. [Extraction]
Add 1.5 mL ice cold 77 % ethanol. Gently dislodge pellet from bottom of tube, and pour etOH + pellet into new 1.5 mL tube. Leave in fridge for 20 min.
?. [Extraction]
Discard supernatant, pipette off residual isOH, and allow pellet to air-dry on bench (take care not to over-dry).
?. [Extraction]
Centrifuge tubes at 3,000 x g for 30 min.
?. [Extraction]
Leave tubes in fridge overnight.
?. [Extraction]
Transfer water phase to fresh tube, record the volumes transferred, and add 1 volume room temperature isopropanol. Mix by inversion for 2 min.
?. [Extraction]
Centrifuge tubes at 3000 x g for 20 min.
?. [Extraction]
Add 1 volume of chloroform:isoamyl alcohol and mix by inversion for 2 min.
?. [Extraction]
Add 5μL of RNAse A (10 mg/mL) and incubate at 37°C for 15 minutes, gently inverting the tubes periodically.
?. [Extraction]
Transfer water phase to fresh tube, record the volume transferred.
?. [Extraction]
Spin at 3000 x g RT for 20 min.
?. [Extraction]
Add 6 mL of chloroform:isoamyl alcohol (24:1) and mix by inversion for 2 min.
?. [Extraction]
Cool down tubes for at least 5 minutes.
?. [Extraction]
Incubate tubes at 65°C for 1 to 1.5 hours, invert tubes every 10-20 min.
?. [Extraction]
Add 30 uL of proteinase K (20mg/mL) to each tube and mix by inversion.
?. [Extraction]
Add 6mL pre-warmed extraction buffer containing β-me to each tube, and resuspend pellets by inverting/tapping tubes.
?. [Extraction]
Centrifuge tubes at 3,000 x g for 5 min. Discard supernatant.
?. [Extraction]
Add 10mL sorbitol wash containing β-me to each tube and mix by inversion for 2 min.
?. [Extraction]
Grind ~2 g leaf material in mortar and pestle under liquid nitrogen to fine powder. Make sure material does not thaw. Transfer to 15 mL tubes.