more novozyme problems

Lisa Vaillancourt lvaillan at MOOSE.UVM.EDU
Wed Mar 9 15:27:59 EST 1994


I hope somebody out there can help, lately we have been having a very hard
time making transformable protoplasts of Schizophyllum commune using our
normal novozyme protocol.  We recently purchased novozyme 234 batch PPM
4356 from Interspex.  It worked OK for one of our strains when used at a
very low concentration for a shorter period of time than normal, but since
then we have not had any luck.  It seems that if we use it at a
concentation high enough to get protoplasts (between 5 and 10 mgs/ml final
concentration) the protoplasts do not transform.  However, if we use less
(between 0.5 and 5 mgs/ml) the digestion is really poor and we get few or
no protoplasts.  Our protocol calls for digestion of chopped mycelium in
buffered 1M MgCl2 at 30C for about 5 or 6 hours, followed by mixing with an
equal volume of water, thus making the concentration 0.5M MgCl2, followed
by spinning relatively slowly to pellet the larger pieces of mycelial
debris, followed by adding an equal volume of 1M sorbitol and spinning
harder to bring down the protoplasts.  When we look at the mycelium after
digestion with the lower concentrations of novozyme, it still looks mostly
intact and there are few free protoplasts, but there are many visible still
trapped in what's left of the hyphae.  If we use higher concentrations, so
that there are more protoplasts, they transform very poorly, though they
look fine and usually regenerate well.  HELP!!  We have been told that this
novozyme does better at 37C than at 30, and that it doesn't work well in
magnesium..and what is the opinion of the usefulness of including beta
mercaptoethanol in the digestion mix??  We are planning to do a
troubleshooting experiment, can anyone give us other ideas to try??  We
would really appreciate it.  





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