Hi,
I have been trying to clone and express my protein with pichia. And the
cloning and transformation works very well. Now I met a problem, the
positive transfromants refuse to grow in BMGY, while the control provided by
invetrogen grows well. So I figured there may be something wrong with my
thing but I don't know where I did wrong. I transform the vector into pichia
by generating spheroplast and I got a few thousand colonies on each plates.
Then I picked up 50 from each plate and screen on MM and MD. They looked
fine on both plates, appearing after overnight incubation at 30 degrees. I
did colony PCR with my primers, and all of them show product of correct
size. Then I thought it was time to proceed to expression, but they didn't
grow. Does anyone have any idea of what was happening here, contamination?
something wrong with the midium or integration? Thanks a lot, please save
me.
whynotwork
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