DNA Extraction S. commune Phenol Chlorophorm PEG

DNA extraction for Schizophyllum commune as described in van Peer et al. 2009 and Ohm et al 2010 with modifications for growth and washing and especially for precipitation of DNA using PEG-8000 as described in huang et al 2016.

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 and place in 2ml tubes.
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

Freeze dry the mycelium

• Contact the proteomics facility to request use of their freeze dryer (mail to dr. Martin Lehmann). Might be in use, so plan ahead!
• In a plastic freezer box, place the samples to be dried.
• Go to the facility (Room G02.035; last lab on right side of right corridor).
• Open each tube and place a little piece of mycelium in it.
• Have them turn on the machine and run it overnight.
• Next day go pick it up.
• Mycelium can be stored at -20°C before extraction.

DNA extraction

• Homogenize the mycelium using the TissueLyzer (located in pre-PCR lab)
  • Add a metal bead to each tube
  • Place the tubes+bead into an adapter
  • Place both adapters with the tubes in them for >2h in a -80°C freezer
  • Take them out (careful... cold ;)
  • Homogenize for 20 seconds at 20Hz
• Use all of the homogenized mycelium
  • add 0.9 ml of extraction buffer
  • and 5 μl RNase (QIAgen in door fridge 1)
• inoculate at 65°C for 15 min

From here work in flow hood!

• Add 0.4 ml of phenol:chloroform:isoamyl alcohol (25:24:1)
• Vortex shortly (shorter the better!) 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 (again, shorter the better!) 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 1 volume of fridge temperature 20% PEG-8000 with 1.2 M NaCl (door Fridge 2).
• Turn the tube until the two liquids mix (PEG is very viscous so might take some time. Do not vortex!)

If there is a visible white cloudy something floating in the liquid:

• awesome! that's all DNA!
• Prepare 2 tubes with 0.5ml 70% ethanol and one empty tube.
• Take a tip and fish the DNA out of the tube with PEG etc..
• Add it into the first tube with 0.5 ml 70% ethanol, let it sit for a bit and then fish the DNA out of this mix again.
• Repeat with the second tube with 70% ethanol.
• Take the DNA and add it in the last tube.
• Dry it in the flow hood until dry.
• Dissolve the DNA in 100 μl ddWater overnight at 4°C

If there is no clear precipitate, continue as follows:

• Precipitate the nucleic acids by centrifugation at 10,000 xg for 10 min at 4°C in the Cooling centrifuge.
• Remove the supernatant by aspiration.
• Rinse the pellet with 500 μL of 70% ethanol.
• Centrifuge the tubes at 10,000 xg for 1 min at room temperature in a microfuge.
• Rinse the pellet once more with 500 μL 70% ethanol.
• Centrifuge again at 10,000 xg for 1 min at room temperature.
• Remove the supernatant by aspiration
• Allow the pellet to dry in the air for 30 min – 2 hours.
• Elute the pellet in 40 μL of DNase-free H₂O.

Test DNA 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

20% PEG-8000 precipitation solution (with 1.2 M NaCl)

For 100 ml:

• 20 g PEG-8000
• 7 g NaCl
• fill to 100ml with demiwater

Store at 4°C