DNA Extraction Phenol Chlorophorm S. commune

DNA extraction for Schizophyllum commune as described in van Peer et al. 2009 and Ohm et al 2010 with minor modifications for growth and washing.

The heavy molecular weight molecules that should be obtained by this method can be further purified by cleaning them on a column if required.

Strain cultivation

Grow the Schizophyllum commune strain in MSMM (a modified version of SMM in which glucose is replaced by 4% w/v Glycerol.

1. When using plates:
   • Pour 90mm plates with MSMM with 2% agar
   • Place a sterile cellulose or (polycarbonate membrane (0.1 μm pore size)) on the plate using sterile techniques
   • Inoculate the plate with 7 little pieces of mycelium
   • Incubate at 25°C for 3-5 days till the plate is covered
   • Remove the little pieces of agar.
   • Harvest the mycelium by scraping it off the membrane with a spatula or knife
2. When using liquid:
   • Fill 100ml MSMM in a Erlenmeyer flask
   • Add ~15-50 little pieces of mycelium to the liquid
   • incubate in a shaker at 25°C for 3-4 days
   • Harvest the mycelium by pouring over a paper filter and rinsing with water
   • fold over filter and squeeze out remaining liquid

Mycelium can be stored at -20°C before extraction.

DNA extraction

• Grind the mycelium in liquid nitrogen using a mortar
• Add more liquid nitrogen and keep grinding till a fine powder is obtained
• Use ~200-300 mg of homogenized mycelium
  • add 0.9 ml of extraction buffer
  • and 5 μl RNase
• inoculate at 65°C for 15 min
• Add 0.4 ml of phenol:chloroform:isoamyl alcohol (25:24:1)
• Vortex shortly to get a cloudy mixture
• Separate the liquid phases by centrifugation for 10 min at 13,000xg.
• Carefully transfer the upper phase (without disturbing the interphase) to a fresh tube.
• Add 0.5 ml of chloroform:isoamyl alcohol (24:1).
• Vortex shortly to get a cloudy mixture
• Separate the liquid phases by centrifugation for 10 min at 13,000xg.
• Carefully transfer the upper phase (without disturbing the interphase) to a fresh tube.
• Precipitate the DNA with 0.8 volume of chilled isopropanol.
• Pellet at 13,000xg for 10 min at 4°C.
• Remove supernatant. If there is a snotty pellet do not throw that away! It contains the DNA. In that case stop here and continue with a cleanup using a column.

If there is a visible pellet:

• Carefully add 0.5 ml 70% ethanol and remove with a pipette
• Repeat the wash step 2X more: add 0.5 ml 70% ethanol and remove with a pipette
• dry the DNA overnight
• Dissolve the DNA in 100 μl ddWater
• Test for
  • integrity on gel,
  • for purity by nanodrop and
  • for concentration using Qubit.

Extraction buffer

For 100ml:

• 2.0 g sodium dodecyl sulfate
• 2.4 g 4-aminosalycilic acid
• 20ml 5X RNB

5X RNase Buffer (RNB)

For 100ml:

• 12.1 g Tris-HCl
• 7.3 g NaCl
• 9.51 g EGTA

Set to pH 8.5