Hello all. I transformed some Agrobacterium competent cells by electroporation and performed colony PCR to see the positive transformants. However, I performed the glycerol stock after 10 days of streaking the cells in a new plate for colony PCR. This plate was stored at -4 degrees. I know that I should make the glycerol stock as soon as I confirmed the transformants, but do you think my cells lost viability during 10 days in the plate before going to -80°C? Thanks
It is generally not recommended to make a glycerol stock directly from Agrobacterium-transformed cells grown on a 10-day-old plate. The search results indicate that glycerol stocks are typically prepared from fresh overnight liquid cultures of Agrobacterium. Storing cells from an old plate may result in reduced viability and transformation efficiency when the stock is later used.
I would suggest to make glycerol stock from a pure and recently grown plate.. so you may use it later to make liquid broth directly from glycerol stock..
My advisor used to say "you can't rush quality". Use your streaked plate to start a fresh liquid culture with selective media & then make your glycerol stock.
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