I am doing Chromatin fractionation assay in the fission yeast Schizosaccharomyces Pombe, and the first step is getting the decent spheroplasts by treating the cells with Zymolyase 20T. I need to do this assay for different HA-tagged strains, and I realized that the time required for the spheroplasting varies greatly from strain to strain, some might need up to 1hr, while the others only about 30 min or even less. Is it the possible reason that people never mentioned about the time required for this step in the protocol? How can I ensure that I have achieved precise spheroplasting and can proceed with the following steps? I usually monitor the cell refringe disappearance under microscope, and also check the refractivity using the spectrophotometer; however, these methods are also not that robust to me, as the spheroplasting can be under- or over-estimated. Thanks a lot!
Catherine
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