TITLE: High molecular weight DNA extraction for long read sequencing
AUTHORS: Karen Cristine Gonçalves dos Santos, Megan McDonald

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Adaptation of the protocol </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">dx.doi.org/10.17504/protocols.io.k6qczdw</span></a></div><div class = "text-block"><span style = ":UNDERLINE;">Optimized for DNA extration from Bipolaris sorokiniana spores.</span></div></div>

[STEPS]
?. [Extraction]
2.5 volume of Buffer A 5 mL2.5 volume of Buffer B 5 mL1.0 volume of Buffer C 2 mLPVP 40 10%                 1mLPVP 10 10%                 1mL
This protocol is an adaptation from https://www.protocols.io/view/high-quality-dna-from-fungi-for-long-read-sequenci-k6qczdw
?. [Extraction]
Grind tissue/spores with liquid nitrogen in a mortar with sand, use 1g of sand per 100mg of starting material. Grind for 2 mins in 4x 15 sec bursts adding liquid nitrogen after each 15 sec grinding burst.
Using a sharp scalpal, I scrape the mycelia and the spores from two fully covered petri dishes (~90mm diameter).
?. [Extraction]
Transfer ground tissue with sand into 50mL Falcon containing lysis buffer. Add 10 uL of RNAse A and 100 uL of Proteinase K and mix by inversion until no clumps are visible. Incubate in rotator at 30 rpm for 30 min at RT.
?. [Extraction]
Cool in ice for 5 minutes
?. [Extraction]
Add 2.8 mL (0.2 vol) of KAc 5M, mix by inversion, incubate on ice for max 5 mins
?. [Extraction]
Spin at 4°C and 5000g for 12 mins
?. [Extraction]
Spin at 4°C and 5000g for 12 mins
With this fungus, there is always an interphase with debris. I take most of the supernatant without taking the phenol phase. Even if there is still debris at the end of the purification, the AMpure beads will not bind the debris, so to increase your final DNA yield it is better to take as much as the supernatant as possible.
?. [Extraction]
Transfer supernatant to fresh Falcon tube containing 17 mL (~1vol) (P:C:I) and mix by inversion.
?. [Extraction]
[Repeat Phenol:Chloroform wash]
Debris is mostly at the interphase, but it also attaches to the tube wall, so it is carried over to the next step.
?. [Extraction]
Transfer supernatant to a new 50 mL falcon and add 10 µL of RNAse A. Incubate in rotator at 30 rpm for 30 min at RT.
?. [DNA precipitation]
Transfer supernatant to a new 50 mL falcon and add 0.1 vol (~1.5 mL) of NaAc and mix by inversion.
?. [DNA precipitation]
Add 1 vol (~ 17 mL) of cold isopropanol and mix well by inversion.
?. [DNA precipitation]
Spin at 4°C and 8000g for 30 mins.
?. [DNA precipitation]
Pour off supernatant, keeping around 1 mL. Use it to dislodge the pellet and transfer it to a 1.5 mL tube.
?. [DNA precipitation]
Spin in a microcentrifuge at 15 000g for 3 min.
?. [DNA precipitation]
Discard supernatant and add 1 mL of cold 70% ethanol, flick the tube to help wash the pellet and spin in a microcentrifuge at 15 000g for 3 min.
[Repeat 2 to 3 times]
?. [DNA precipitation]
Discard the supernatant, spin again to get the remaining ethanol out. Air dry the pellet for 1 min.
?. [Re-dissolve DNA]
Add 200 μL of 10mM Tris pH 8.5 and flick the tube to dislodge the pellet.Let it dissolve over night.Measure concentration with BR Qubit and absorbance with Nanodrop.
ABCDEF1Sample*Qubit (ng/µl)Nanodrop (ng/µl)A260/A230A260/A280Total amount (µg)2WAI241172.919232.0222.16114.583WAI338289.423532.0062.12917.88
?. [AMPure bead cleanup to remove small DNA Fragments]
Vortex AMPure beads and add 0.6 vol of beads to the DNA sample. Mix beads and DNA by flicking the tube.DO NOT VORTEX BEAD/DNA MIXTUREIncubate in a rotator 30 rpm for 10min
?. [AMPure bead cleanup to remove small DNA Fragments]
Spin the beads/DNA mix for 1-2 sec in a microcentrifuge or table top centrifuge. Place the tube on a magnetic rack. Wait for the beads to pellet against the magnet.Slowly pipet off the supernatant.
DNA should now be bound to the AMPure beads, however I often save this supernatant, just in case something goes wrong. If your final elution contains little or no DNA, you can come back to this supernatant and "rescue" the DNA from there by adding an equal volume of beads. If all goes well this supernatant is waste that can be thrown away at the end of the extraction
?. [AMPure bead cleanup to remove small DNA Fragments]
Pipette off ethanol and repeat wash.
?. [AMPure bead cleanup to remove small DNA Fragments]
Remove from magnet and spin the beads for 1-2 sec in a microcentrifuge or table top centrifuge.  Place back on the magnet and let the bead pellet stick. (Should be very quick 1-2 secs).Pipette off the remaining ethanol. Do NOT let the ethanol dry.
?. [AMPure bead cleanup to remove small DNA Fragments]
Take the tube from the rack and add 49 µL of nuclease-free water and flick the tube to ressuspend the beads. Incubate at 37°C for 5 min.
?. [Extraction]
Add 100 µL of Proteinase K and incubate in rotator at 30 rpm for 30 min at RT.
?. [AMPure bead cleanup to remove small DNA Fragments]
Spin the tube and place it at the magnetic rack. Wait for the beads to pellet and recover supernatant.
Measure concentration with BR Qubit and absorbance with Nanodrop.
At this step, the ratio A260/A230 falls abruptly, but sequencing occurs without any issue.
Repeat clean up if you loose more than 30% of your DNA.
From here we follow with Oxford Nanopore protocol "1D genomic DNA by ligation (SQK-LSK108)", version GDE_9002_v108_revU_10Oct2016. For the elution, at each step in the library preparation, we incubate the beads with water/elution buffer at 37°C (5 minutes instead of 2 at the DNA repair and the End-prep steps).
?. [AMPure bead cleanup to remove small DNA Fragments]
Wash beads with fresh cold 70% ethanol,  using enough volume to fill the tube. Pipette the ethanol in the tube on the wall opposite of the bead pellet.
?. [Extraction]
Add 1 vol (17mL) of P:C:I, mix by inversion and spin at 4°C and 5000g for 12 minsSolution will look milky like this.After centrifugation, there should be little to no debris in the aqueous layer, as in here.