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[mbsea]

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...

[sd_labber]

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.