TITLE: Modified HMW DNA Isolation from Stramenopiles with Agar Plugs
AUTHORS: Vincent Bielinski, Chris Dupont

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is the adaptation of previously published methods for use in isolating intact high molecular weight DNA from stramenopiles for long-read sequencing.  The advantages here are minimal amount of centrifugation steps on isolated DNA, as well as the long-term storage of the DNA while in plugs to allow experimental flexibility.</div></div>

[STEPS]
?. Add 0.5 g of LMP agarose to 25 mL TAE (2% final)
?. Microwave for 30 secs or until solution begins to bubble
?. Repeat heating steps as necessary (2-3x)
?. Move flask to 50oC air incubator, swirling a few times to bring agarose into solution
?. Thaw 50 mL conical vial with cell pellets on ice (cell pellet ~1.5g for this expt)
?. Add 50 mL 1M sorbitol and resuspend pellet gently
?. Let cells sit on ice 1 min
?. Harvest pellet with 10 min spin at 3000 x g at 4oC
?. Remove sorbitol wash supernatant
?. Resuspend cells in 5 mL protoplasting solution
?. Incubate cell suspension at 37oC for 10 min, then remove from water bath
?. Keeping agarose warm, mix 5 mL of 2% agarose with cell suspension
?. Pipette 100 uL of cell-agar solution into disposable plug molds (Biorad #170-3713)Note:  Wide-bore tips can make it easier to dispense agar-cell solution into the molds if regular tips keep clogging
?. Place molds at 4oC for 15-30 min to solidify
?. Once solid, transfer plugs to new 50 mL conical vialNote: The plug molds come with a comb for pushing the plugs out into the new conical vial
?. Wash plugs with 25 mL of wash buffer (30 min at RT with soft shaking), the tube can be taped on its side on an orbital rocker and mixed slowly
?. Add 10-20 mL of proteinase K solutionNote:  This enzyme can be expensive, so scale volumes accordingly.  You really just need enough to cover the plugs.
?. Incubate for 24 hrs at 50oCNote:  At this point you should see cell lysis and your solution change colors.  For diatoms, the solution becomes green within 30 minutes, then changes to brown over the 24 hrs.
?. Wash plugs 2x with 25 mL of wash buffer (30 min at RT)
?. Add 10-20 mL of Pronase solution to plugsNote:  This step can be omitted if you are confident that the Proteinase K treatment is sufficient for your organism, but will not harm your DNA if left in.
?. Incubate plugs in Pronase solution for 4 hrs at 37oC
?. Wash plugs 2x with 25 mL wash buffer (30 min at RT)
?. Store at 4oC or proceed to next stepNote: Plugs can be left in wash buffer after this step for up to 1 month at 4oC
?. For agarose digestion, we often process a small amount of the total plugs to first determine DNA yield and often this small number of plugs (~5) can yield enough DNA for a Nanopore run
?. To digest, remove 3-4 plugs (or more) from conical vial and transfer into new 1.5 mL Eppendorf tube
?. Wash 2x with 500 uL of 1x beta-agarase wash buffer (NEB) on ice for 30 min each cycleNote:  the NEB buffer is composed of 10 mM Bis-Tris-HCl, 1 mM EDTA, pH 6.5@25°C if you want to make it in-house
?. Remove excess wash buffer from tube with pipette
?. Heat tube at 65oC for 10 min in heating block
?. Cool block and solution to 42oC Note:  Leave to cool at least 10 min, as agarase gets inactivated over 45oC
?. Add 1U Beta-agarase per 100 uL melted agarose volume
?. Incubate for 1-2 hr at 42oCNote: Tubes can be gently shaken during treatment, but leaving on bench and mixing every 30 min should work as well
?. Allow tube to cool to RT
?. Store at 4oC overnight or move on to extraction (below)
?. Phenol extraction for HMW DNANote: Wear proper PPE and work with phenol in a fume hood as much as possible
?. Mix 250 uL of liquefied agar solution with 250 uL of Tris-buffered phenol
?. Rock end over end slowly for 10 min at RT	Note: Extractions can go for up to an hour at RT if greater DNA yield is desired
?. Separate phases by centrifuging for 10 min at 16,100 x g
?. Transfer aqueous layer (around 250 ul) to new tube
?. Add 25 ul of 3M NaOAC to tube	Note: We’ve noticed that addition of the 3M NaOAc causes the aqueous layer to become turbid and opaque, so if you see this it’s not unusual
?. Invert tube slowly 10 times until mixed
?. Add 825 uL of 100% EtOH
?. Invert tube slowly 10 times until mixed
?. Allow to sit at -20oC for 1 hr minimum, overnight precipitations may increase DNA yield
?. Centrifuge for 20 min at 16,100 x g at RT	Note:  make sure to have the centrifuge set to lowest acceleration/deceleration speed possible if you want to avoid shearing
?. Remove supernatant gently with pipet
?. Wash pellet quickly in 500 uL 70% EtOH + 50mM CaCl2Note:  We have found adding calcium can help precipitate out carbohydrates or lipids often associated with the gDNA
?. Centrifuge for 5 min at 16,100 x g at RT
?. Repeat 70% EtOH + 50 mM CaCl2 wash
?. Centrifuge for 5 min at 16,100 x g at RT
?. Remove wash solution with pipette
?. Allow pellet to air dry for 5-10 min at RT
?. Resuspend pellet gently in 50-100 uL TE, pH 8.0 - DO NOT MIX WITH PIPETTENote:  If you have trouble resuspending your pellet, you can either leave it at 4oC overnight or heat the tube to 37oC for about 10 minutes to help solubilize DNA
?. Analyze 5 ul of resuspended pellet on 0.8% agarose gel to determine DNA quality, using wide-bore tips to transfer DNA solution to gel while preventing shearingNote:  We currently use Qubit quantitation for measuring the concentration of HMW DNA