Hi all,
I just wanted to know the time required for transformed pichia to grow on MD plates til the colony is visible. I have transformed with my gene of interest into to Pichia pastoris(1*109 order cell) with 150 ng of DNA(in pPIC9 vector linearized with sacl) .As this is my first attempt,I do not have any previous experience on it. I used 1500V,25 MicroF,200 Ohm and 4.2 milliseconds for the electroporation experiment.
And I plated with 100 microlitre .One literature says it is 2 days and other is saying 4-6 days.Would you guys share your experience? Size of the inserti is around 780bp.
Hello i am excited to know that you are working with Pichia. But i need little detail to answer your question.
1. What was the O.D at which you transformed your cells?
2. Which and how much antibiotic did you use in plates?
3. Temperature of incubator you have placed your plates in?
I used OD 3.7
I did not use any antibody because I use His4 gene as selection marker. After transformation, I incubated the plates at 28 degrees for 3 days.
My experiments worked perfectly fine. I just had few doubt which are still unfathomable to me.
It depends on the exact strain, the growth medium and of course, incubation temperature. At the very minimum, by day 3 post-plating you should see tiny colonies coming up.
colony are very small and look similar like DH5 alpha ....it is initially transpereant and basic staining canh be used to see budding behaviour
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