TITLE: Pleurozium schreberi (moss) DNA extraction
AUTHORS: Eric Pederson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a detailed protocol concerning the DNA extraction from the genome paper titled "</span><span style = "font-weight:bold;">Genome Sequencing of</span><span style = "font-weight:bold;font-weight:bold;font-style:italic;"> </span><span style = "font-weight:bold;font-style:italic;">Pleurozium schreberi</span><span style = "font-weight:bold;">: The Assembled and Annotated Draft Genome of a Pleurocarpous Feather Moss</span><span>" (https://doi.org/10.1534/g3.119.400279).</span></div></div>

[STEPS]
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Ensure that the sterile bench is clean off and all the lab plastic and equipment is sprayed and placed in the sterile bench for at least 15 minutes.
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Grind the tissue using mortar and pestle in the presence of liquid nitrogen until finely ground. Transfer frozen ground tissue to a 2 ml tubes.
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For every gram fresh weight add;- 1 mL CTAB buffer- 300 μL N-lauroylsarcosine buffer- 60 μL Proteinase K stock- 90 μL Pronase stock - 120 μL β-Mercaptoethanol.
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Mix by inversion and incubate in a water bath in 60°C for 60 min.
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Cool to room temperature.
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Clean forceps with ethanol and burn until red.Proceed to take the tissue and place in 2 ml tubes for storage or to use immediately.
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Pre-warm the CTAB buffer and N-lauroylsarcosine buffer.
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Add 1 vol of sterile 5 M ammonium acetate, mix, and incubate for 5 min.
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Centrifuge for 10 min at 15,000 g at room temperature.
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Precipitate the DNA from the supernatant with 1 vol of isopropanol at -20°C for 30 min.
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Weigh the tissue by subtracting the tube.
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Centrifuge for 30 min at 12,000 g at 4°C.
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Wash pellet with 70% ethanol, dry, and redissolve in 1 mL TE buffer.
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If the DNA will be used for sequencing, use water instead (milli-Q water)
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Add 100 μL of RNAse stock. Incubate at 37°C for 15 min.
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Perform 2 extractions with 1 vol of chloro-form-isoamylalcohol (24:1).
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Precipitate the DNA from the aqueous phase with 1 volume of isopropanol.
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Centrifuge for 30 min at 12,000 g at 4°C.
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Wash pellet with 70% ethanol
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Air dry to ensure ethanol is gone.Do not overdry
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Redissolve the pellet in 500 μL TE buffer.
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Vortex for 1 minute and centrifuge for 10 min at 12,000 g at 4°C.
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If the DNA will be used for sequencing, use water instead (milli-Q water)
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Use pipette to discard the ethanol
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Transfer the upper, aqueous phase to a fresh tube and add 1 volume of chloroform:isoamyl alcohol (24:1).
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Vortex for 1 minute and centrifuge for 10 min at 12,000 g at 4°C.
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Transfer the upper, aqueous phase to a fresh tube.
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Remove any transferred chloroform by centrifugation 10 seconds at top speed followed by removal of the bottom phase.