Hello.
I am working on the generation of C. acutatum protoplast from young mycelia.
I used drisease as a lysing enzyme in 1 M NH4Cl.
It seems to work well, but the protoplasts generated tend to bind to
hyphal fragments, so I get only the small number of protoplasts.... virtually..
How can I separate the protoplasts from hyphal fragments?
I think you need to treat some reagent to protect hyphal wall regeneration or to increase the concentration of lysing enzyme and other enzymes for digesting cell wall. Because usually cell wall can be also recovered during enzyme incubation, although enzyme is working. If you get a few protoplasts as bursting, there may be also osomosensitive problem.
Do you have this paper (see attached). In the paper, there is one paragraph dedicated to the production of C. acutatum protoplasts in detail:
"After a 2-h digestion, the solution was passed through cheesecloth and glass wool in a 50-ml syringe. Fungal protoplasts were harvested by centrifugation at 65000 x g for 10 min, and washed with STC buffer (1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl2)."
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