pichia epression problem - (Aug/06/2012 )

Hi everybody, I am trying to express my betaglucosidase gene in pichia but i have a question i inoulated a single colony in 5 mL mgy medium 300 rpm 28 C the next day i inoculated the whole culture to 20 ml MGY buffer (total 25 mL). 24 h later the od could only reach 1.3. So i santrifuge the cells 3000 rpm 5 min and resuspend them in 20 mL fresh mgy buffer and 300 rpm 28 C an overnight again. The od is noq around 2. My question is do you think i miss the log phase of the cells because of 2 overnight waiting? Could anyone offer me a pichia expression protocol because i am trying to do what invitrogen says; i could not catch the od 600.

best regards...

I had the same problem and it turned out to be a transformation issue (we were using an old electroporator). Try starting fresh with your transformations and pick the most robust (biggest colonies) from your YPDS plates to inoculate in MGY O/N. We picked colonies after 4 day incubations at 30 degrees.