Greater numbers of protoplasts have been obtained by adding 0.1 N NaOH to the mannitol solution to obtain pH 6-7. One drop of NaOH should be sufficient to adjust the pH of 50 ml of solution. Autoclave or otherwise sterilize the solution in 100 ml volumes. MES is a pH buffer. The two enzymes can be mixed.
Students add enzymes to buffer solution immediately before use.
These enzymes are not stable at high temperatures or for long periods of time. Cellulysin is Sigma's proprietary name for a cellulase isolated from Trichoderma uride. This enzyme degrades cell walls. The procedure and analysis questions found on the student page can stand alone or can be embedded within any context provided by the teacher. The following outline uses the problem of transformation as the context for the activity.