I used this protocol successfully modified an unpopular fungus.

1. Grow your fungi in 1L flask cantaining 100 ml suitable media (such as YPD) in a controlled temperature and shaking to a log phase.

2. Harvest the culture by filtration through sterile Miracloth and wash mycelia with sterile water then 0.5 M MgSO4. dry the material by squeeze with dry tissue.

3. the mycelia transfer to a 50 ml conical tube, add 30 ml of OM buffer (1.2 M MgSO4, 10mM NaPO4, pH5.8, 1.2m MgSO4-10mM-Na2HPO4(not completely disolved) dilute with 1.2m MgSO4-10mM-NaH2PO4 into pH5.8-autoclave), fully suspend first. then add 300 mg Glucanex (sigma) in 10 ml OM buffer, filter sterile and add to the tube. incubate at 30C for 3 hours with 100 rpm shaking (check undermicroscope before go to next step).

(with 500mg (some use 200 mg) Glucanex (Novo Ferment, Basel, Switzerland), shake gently (incubate at 30C with 75 rpm for 2-3 hours) to dispers hyphal clumps.) at this time point, cut linearize the plasmid or get the transformation PCR product ready for homologus recombination with G418 as selection marker. You need to test your fungus wont grow on G418 plate as wild type. Or a known plasmid with what you want which can be used for your fungus.

4. cool the centrifuge and STC buffer(1.2 M sorbitol, 10 mM Tris-HCl pH7.5, 10 mM CaCl2). check the protoplast on microscope. if ready, then filter through 3 layer of miracloth (sterile), add at least 1 vol of STC buffer. mix gentlely. put on ice. spin down at 2000 g (2700rpm in the big machine) at 4C. discard the supernatant.

5. handle gently, wash the protoplasts twice by filling the tube with STC buffer and resuspending with about 2 ml. And pellet by centrifuge for 10 min at 1240g at 4C as in step 4.

6. resuspend the protoplasts in 1 ml of STC and count microscopically, Yield should be in the range of 2-5X108.(one in a small square equal 2.5X 10to the power of 5. normally you need to dilute 10-100 time to count)

7. Directly use107 protoplasts (100-150 ul) for the transformation.  Or aliquot 107 protoplasts (100-150 ul) into 1.5 ml eppendorf tubes, (someone use cool slowly to -70c then store)snap frozed in liquid nitrogen and store at -80C.

8. Add 2-3 ug linearised DNA in a total volume of 150 ul STC. Mix gently and incubate at RT for 15-25 min.

9. add 1 ml PTC (60% PEG 4000-(sigma) 10 mM Tris-HCl pH7.5, 10 mM CaCl2-filter sterilized), gradually by 2-3 adding (not put in with one go), mix gently by inversing and incubate at RT for 15-20 min.

10. Add protoplasts to 125-150 ml melted (50C) YPD agar,  mix gently and pour into 5-6 plates. Incubate for at least over night  in 37C. then overlay with approx 15 ml top agar (YPD agar containing 300ug/ml G418).

11. incubate until you can see colonies appear. check daily.

12, genetic verify the target modification.