The pichia pastoris was GS115, and the vector was pPIC9K. The previous steps including electrotransformation, His- plate selection and G418 screen were normal. However, when I ready to express the protein in BMMY, some transformants (about 1/2 of all) cannot growth in BMGY. These transformants growth well in YPD liquid and look smaller in YPD plate than normal transformants. So who know why these transformants cannot use glycerol as carbon resource? Is there any gene mising?
Have you checked those smaller colonies to ensure they are not contaminants?
Did you check with colony PCR, your gene of interest inserted in which location. Find the colony morphology of growing culture in YPG liquid and solid Media.
What I do for colony PCR of Pichia is to take a tiny portion of the colony (we are talking less than approx. 0.5 mm2) with a sterile tip, resuspend it in 20 uL of 20 mM NaOH and incubate it at 98*C for 45 min. Then I spin the sample at 10,000 x g for about 5 min. and use 1 uL of the supernatant in the PCR reaction. Don't remember where I stole it from, but has always worked.
Don't forget to include a positive control, by the way.
Hope it helps!
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