DNA Extraction Phenol Chlorophorm S. commune

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DNA extraction for Schizophyllum commune as described in van Peer et al. 2009 [1] and Ohm et al 2010[2] 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[2]).

  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
  • 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

References:

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