DNA Extraction S. commune Phenol Chlorophorm PEG

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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 modifications for growth and washing and especially for precipitation of DNA using PEG-8000 as described in .

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 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
  • 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

References:

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