question about transformation - (Aug/20/2012 )

Hi

Can any one please help me out with the transformation of Pichia pastoris?

We are trying to express gp96 (glyco protein 96) mutants in pichia pastoris.

I have tried electroporation as well as CaCl2 treatment for the production of competent cells.

But it was of no use

After electroporation, I am adding 1mL of ice cold sterile 1M sorbitol and then incubating them for approximately 3 hours at 30^o C and plating them on YPDS + Zeocin plates

I have also tried other methods like addition of 0.5mL sorbitol and 0.5mL of YPD media to the cells immediately after electroporation and then incubate them for 3 hours at 30^o C.

Please send me your suggestions so that I can make it work.

Thank you.

Check that your zeocin is the appropriate concentration - titrate the level needed for the cell strain you are working with.