Hi all,
First time working with yeast and I have some questions. Following electroporation (1800V) of my linearized pPIC9K plasmid into GS115 I grew them up on MD agar as per the Invitrogen Pichia Expession manual. However, after 2 days of growth I thought the colonies looked odd compared to their usual morphology on YPD agar. They were almost furry with lots filaments viable. After a few days at 4 degrees small blue/black spots appeared and so I assumed there was contamination..... plated the original samples again on YPD plates to see if there was normal growth and they still look different to my control GS115 plates, with some of the colonie having a pink colour to them. Not sure if the morphology is supposed to change after electroporation or if something else is going on...
Can anyone advise on this? Any help or suggestions would be greatly appreciated!
The GS115 morphology should look the same (white, roundish colonies that smell like yeast) after transformation/electroporation compared to GS115 grown on YPD. Although the colonies grown on MD agar will take longer to appear compared to YPD. Somewhere along the line you picked up contamination. Be sure to autoclave your reagents longer than recommended to ensure sterility.
2/15/22
Dear Laura,
I agree w/ Tom Masi's answer. From the descriptions you gave, it sounds like your culture has become contaminated. The furry appearance of the colonies, presence of filaments, pink coloration (probably Neurospora) all indicate contamination. Have you checked the cultures under a microscope? That would confirm it. You need to try again w/ media that has been sterilized properly and practice good sterile technique. Good luck!
Bill Colonna Iowa State University, Ames, Iowa [email protected]
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