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pichia epression problem


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#1 mbsea

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Posted 06 August 2012 - 12:46 AM

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...

#2 sd_labber

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Posted 22 August 2012 - 09:12 AM

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.




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