TITLE: Fungal CTAB DNA Extraction 
AUTHORS: Derreck Carter-House, Jason Stajich, Sarah Unruh, Tania Kurbessoian

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is a CTAB DNA extraction method for filamentous fungi. Its purpose is to extract high molecular weight genomic DNA for genome sequencing.</div></div>

[STEPS]
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Each tube of lysis buffer will be split in half so prepare one tube of lysis buffer for two samples.Prepare Lysis Buffer by adding to each 2mL microcentrifuge tube  µL Buffer A,  µL Buffer B,   µL Buffer C,  µL  PVP, and  Proteinase K to microcentrifuge tube, mix, and then split equally into two 2 mL tubes.
650 µl
650 µl
260 µl
175 µl
10 µl
65 degree preheated solutions are much less viscous and allow for better mixing and equal splitting.
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Place in hot plate and heat to .
65 °C
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Take your liquid nitrogen, carefully and completely immerse the inside of your mortar.Add your pestle inside the liquid to super cool it as well.Add your fungi using a spatula cleaned with ethanol, make sure to grab only the fungi and as little agar as possible.Grind fungal tissue in liquid nitrogen, add of tissue to each tube.
100 mg
Take your time on this step by slowly crushing the pieces of frozen fungi. Make sure the mortar stays cold by carefully adding more liquid nitrogen. (Add a little at a time to ensure the crushed fungi remains in the mortar).Grinding can take up to a minute, make sure to put enough force to get a fine powder.
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Incubate  at  mixing by inversion frequently (- min).
65 °C
Sometimes this step can generate gas due to heat expansion. As you are mixing the inversion, make sure to open the cap a bit to relieve the pressure.
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Add  5M Potassium Acetate to each tube, mix by inversion, incubate on ice for .
280 µl
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Add - (as much as the microcentrifuge tube can reasonably hold) Phenol:Choloform:Isoamyl alcohol, 25:24:1 (PCI), mix by inversion (>5 min) or vortex briefly (5-10 seconds) then incubate for 2 min at room temp (RT).
500 µl
700 µl
DO NOT overfill the tube though. The phenol:chloroform:isoamyl alcohol can seep out of the edges and make it very slippery to hold. (Vortexing will be difficult).If you have over-filled, tip out a bit of your solution onto a napkin and throw it away.
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Spin at
Centrifuge: 6000 34, 10 min
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Take supernatant, transfer to new 2 ml microcentrifuge tube add equal volume Choloform:Isoamyl alcohol, 24:1 (CI) (usually about 1000ul).
If you can, use cut pipette tips here to ensure DNA doesn't shred due to the force of pipetting through a small opening.Cut pipette tips must be autoclaved before use.
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Mix by inversion () then incubate at room temperature for
[at least]
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Centrifuge: 6000 34, 10 min
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Take supernatant (usually ) and add it to a new 2 ml tube then: a.	RNAse treatment ( RNAse A, ,  - )b.	Optional 1-2 additional CI washes
700 µl
2.5 µl
37 °C
Additional CI washes will result in cleaner DNA but will also have a lower yield.
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Add 1/10 vol 3M Sodium Acetate, mix, add 1 vol Isopropanol
How to tell what 1/10 volume will be? Check the side of the eppendorf tube with your liquid inside. Is the line at 750uL? Divide this by 10 and that will be the volume of Sodium Acetate you add. (in this case 75uL)
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Gently mix by inversion and incubate at , should start to see lots of DNA threads.
0 Room temperature
This may look like a super sugary solution when you add water to it. It'll have ripples inside. Don't worry if this ends up dissolving after some time.
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pour out the supernatant.
Centrifuge: 3000 34, 2 min
A tip here is to align your tubes all in the same orientation. This will help with clear DNA or very low yields of DNA.The next step requires you to pour our the supernatant. It's good to have a general idea of where the DNA is as you pour out. Make sure to pour slowly and in one step (vs pouring a little and orienting the tube to stand, back and forth which essentially mixes your DNA and dislodges it from the edge of the tube).
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Wash with  freshly prepared, cold
1 mL
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at , pipette out the Remove as much EtOH as possible before drying.
Centrifuge: 3000 34, 2 min
Pour ethanol out slowly and remove excess EtOH.
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Dry pellet at RT for  -  and/or  for  to dry any leftover ethanol OR if you have a vacuum chamber, place your samples there for 10-20 minutes under constant vacuum.a.	Resuspend in  -  TE (adjusted to ) at If DNA still has high protein:Optional CI wash add 600-800 TE buffer at , resuspend DNA, add equal volume CI*, mix as directed in step 9, carry on protocol from there without the RNAse and CI steps.
65 °C
50 µl
100 µl
65 °C
65 °C
*For example, take 500-600 uL supernatant if added 800 uL CI
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Assess DNA quality on NanoDrop.DNA concentration may not be accurate but the absorbance values will determine the purity of the sample.
Remember:Ranges for high quality DNA260:280 - 1.80-2.00260:230 - 2.00-2.20
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Assess DNA concentration with Qubit Fluorometer with DNA Broad Range (BR) assay.
* Dillute   of DNA with water and use all   for the Qubit Assay.
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Assess DNA quality on Agarose gela. Make 0.8- 1% agarose gel with  EtBr solution per 100mL of gelb. Mix equal parts loading dye and DNA sample and load into wells (leaving space for DNA ladder)c. Add equal amount of ladder as loading dye + DNA to the first and last welld. Run the gel (75-120v) for   to   or until samples have run 3/4 of the way through the gele. Visualize bands to assess size of DNA fragments
3 µl
What you are looking for is high quality DNA.Most should stay in the well. What you don't want to see is shearing of DNA, which will have a large blur down your entire well.Also a thing to look for is a general smearing at the bottom of the gel, indicating protein contamination. If you have this, follow Part A in Step 18.
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For reference genome sequencing or other applications where a high volume of DNA is required, tubes of the same sample can be combined. Be sure to check the concentration of the final sample. For applications requiring very clean DNA, it may be helpful to perform additional chloroform:isoamyl washes (Step 12b). As this reduces final yield, it may be helpful to perform multiple DNA extractions of the same organism/sample.
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Heat a water bath to  for use in step 12 of this protocol
37 °C