TITLE: Quick Fungal DNA extraction from colonies on plates
AUTHORS: Johannes Debler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Adapted from </span><a href="https://doi.org/10.5423/PPJ.2009.25.1.108" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Chi, M. H., Park, S. Y., & Lee, Y. H. (2009).</span></a></div><br/><div class = "text-block">Colony PCR on fungal colonies grown on plates does not work as well as it does for bacteria (it usually doesn't work at all). DNA therefore needs to be extracted first. As this DNA will only be used as a PCR template to check for presence / absence of individual genes I am not too concerend about high molecular weight or purity.</div><div class = "text-block">This protocol is quick and can be used to process several samples at the same time. </div></div>

[STEPS]
?. [Make stock solutions]
AB11M TrisHCl pH 8.0&nbsp;100 ml212.14 g&nbsp;Tris base or Trizma base3up to 100 ml&nbsp;deionised H2O4Adjust pHconcentrated HCl  AB12M KCl (Potassium chloride)200 ml215.11 gKCl (Potassium chloride)3up to 200 mldeionised H2O  AB1500 mM NaEDTA (pH 8.0)100 ml218.6 gEDTA disodium salt dihydrate3up to 100 mldeionised H2OAutoclave stock solutions at 121°C
AB11M TrisHCl pH 8.0&nbsp;100 ml212.14 g&nbsp;Tris base or Trizma base3up to 100 ml&nbsp;deionised H2O4Adjust pHconcentrated HCl
AB12M KCl (Potassium chloride)200 ml215.11 gKCl (Potassium chloride)3up to 200 mldeionised H2O
AB1500 mM NaEDTA (pH 8.0)100 ml218.6 gEDTA disodium salt dihydrate3up to 100 mldeionised H2O
?. [Make Extraction Buffer]
AB1Extraction Buffer100 ml22M KCl50 ml31M Tris-HCl pH 8.010 ml4500 mM NaEDTA2 ml5deionised H2Oup to 100 ml
AB1Extraction Buffer100 ml22M KCl50 ml31M Tris-HCl pH 8.010 ml4500 mM NaEDTA2 ml5deionised H2Oup to 100 ml
?. [Preparation]
Per sample prepare 2 x 1.5 ml Eppendorf tubes. Label them with sample number and add:Tube 1: 500 ul Extraction Buffer + 1 stainless steel beadTube 2: 300 ul Isopropanol
?. [Harvest fungal material]
With a sterile toothpick or pipette tip remove a fungal colony or a piece of mycelia of about 3-5 mm x 3-5 mm in size from a plate and put it into Tube 1 (containing the Extraction Buffer and steel bead).
Try not to use a piece that is too big, as that might lead to carryover of too many PCR inhibitors. I usually use my toothpick like a sewing machine needle, punching out a piece of mycelium all the way to the bottom of the agar plate. Then I use the toothpick to slice underneath the actual mycelial mat to try to cut off as much of the agar as possible.
?. [Homogenise fungal material]
Homogenise samples for 5 min at 1500 rpm in the MiniG 1600.
?. [Pellet Cell Debris]
Pellet cell debris by centrifugation at 17.000 x g for 5 min.
?. [DNA Precipitation]
Transfer supernatant to Tube 2 (Containing 300 ul Isopropanol)Vortex for 5 - 10 secondsPellet DNA by centrifugation for 5 min at 17.000 x g
?. [Wash DNA]
Discard supernatantAdd 800 ul 70% EthanolVortex mix sample, make sure pellet comes loose from bottom of the tubePellet DNA by centrifugation for 5 min at 17.000 x g
?. [Dissolve DNA]
Discard supernatantRemove as much of the ethanol as possible by drawing drops out with a pipette tipLet leftover ethanol evaporate for 5-10 minDissolve pellet in 50-100 ul H2O or 1x TE bufferMeasure DNA concentration (e.g. Nanodrop)store DNA at -20°C