Our lab has been having issues digesting yeast cell walls with Zymolyase during cell fractionation. We use cultures with an OD of 0.6-0.7 and have increased the conc. of Zymolyase to 20mg/mL (100uL into 10mL volume of culture) and the incubation time (1hr at 30C). We still are not seeing efficient digest of the cell walls and the last time we performed this protocol, we saw a weird viscous debris in the culture. The buffer we use during digestion has 1M sorbitol, 30mMDTT, 10mMMgCl2, 25mM EDTA and 50mM Tris pH 7.5. Any ideas as to what may be wrong? or what we could do to improve the efficiency?
Hi there,
How do you actually perform lysis and monitor lysis efficiency?
you must also take into account the age of the yeasts, you take yeasts of 24 hours, 48 hours of culture or more or less? more it is young more the wall is young and thus easily digestible by the enzymes
Try also other osmotic stabilizer like MgSo4 intsead sorbitol. you can also increase the incubation time in the presence of the enzyme
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