Hi all,
I am trying to conjugate a plasmid from E.coli ET12567 into anAmycolatopsis strain. The plasmid is integrative so should integrate into the genome of Amycolatopsis.
I have a negative and positive control of just Spores. Negative is overlaid with antibiotics, positive is not.
But when I overlay with antibiotics to select for exconjugants (or kill the spores in case of negative control) I have way more growth after a few days on my negative control as opposed to the actual conjugation plate?
I've attached 2 images to show what I mean - more growth on negative control
I've tried increasing the concentration of antibiotics incrementally from 100ug-300ug/mL, accounting for agar volume.
Any ideas what to do?
I'm thinking of swapping antibiotic resistance markers out but I only have a few months left of my PhD and I'm not sure if there will be a faster or simpler fix?
Thank you so much
Could you be misreading the plates? I presume the one with a few hundred isolated colonies is the negative control, and the other plate is the positive control. It looks to me like that plate is actually a lawn so with millions of colonies and not just a few hundred. The plate is no longer translucent like the space between the colonies on the other plate. But since nothing is labelled I"m not positive which is which.
Michael J. Benedik Hi! Thank you for your response, apologies, the labelling is on the underside of the plate, but you are correct - the one shown with many colonies is the negative control. It hasn't lawned however, this is SFM agar so it is not translucent
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