To cure my Streptomyces strain of a plasmid, I used 5-FC as a negative selection marker. So, cells without the plasmid (which are the ones I'm looking for) will be able to grow on agar plates supplemented with 5-FC.
I'd done a replica plate and found that the colonies I'd picked do not grow on the agar plate supplemented with the given antibiotic (selection marker for said plasmid).
Is it advisable for me to confirm this by growing these cells on plates with antibiotics? This time, I intend to spread a larger amount of cells to confirm that the plasmid is cured. Do you think this is redundant or it's better that I do this confirmation step?
it never hurts to double check especially when using counter selection. Optimally take a pure clone you intend to use and patch in the following order: 5-FC plate, antibiotic, 5-FC+antibiotic, regular growth media. If you are extra paranoid you can add positive controls to each (which should grow on each plate with the exception of 5-FC + antibiotic, nothing should grow on that especially the primary transformants).
Hanna Alalam Ah good point. I'll try that out. Thank you so much!
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