You can try a buffer of 1M sorbitol in 50 mM phosphate buffer at pH 7.5 and add 0.1% final of 2-mecaptoethanol (required to activate the zymolase) as the buffer. You may need to add as much as 1 mg/ml of zymolase. This should result in the production of protoplasts from which you can easily isolate DNA by ETOH precipitation.

Thank you for your help. I will try your suggestion. By the way, wish you have a sucessful new year!

Good luck. What fungi are you doing, it can make a difference on what buffers to use and what enzymes.

I'm doing Penicillium marneffei and detecting it in clinical sample. The concentration of P.m in blood specimens is quite low, around 1 - 100 CFU/ml. Previously, I used lyticase from Sigma. But the result from realtime PCR was really bad and inconsistent. Now I use zymolase prepared in YeaStar DNA kit for clinical samples and lyticase for fungal isolates. I didn't find any problems from the protocol except the enzyme buffer. So I hope to receive the experiences from others.

I would suggest 2 things. First, you might need to use 1.2 M MgSo4 as mycelial fungi do not form protoplasts very well in sugar alcohols like the sorbitol, so if the sorbitol doesn't work try the MgSO4. The advantage is that the protoplasts formed in the MgSO4 may actually band toward the top of the solution when you centrifuge it. Second, if you are doing RT-PCR on blood samples, it would likely be much more effective and faster to just use a mechanical breakage of the samples, like a bead beater and then use a kit for isolating the DNA.