The colonies for innoculation were tested for resistance and they did not grow on amp and kan LB agar plates, but magically somehow the electrocompetent cells made from them are resistant to both the antiobiotics and grew a lawn in 100ug/ml plates. Any suggestions as to where it might have gone wrong?
Maybe this help: http://science.howstuffworks.com/environmental/life/cellular-microscopic/question561.htm
In my opinion I don't share to much time why this happened, I will take new TOP10.
Hi,
Did you test your antibiotics stock solutions? Maybe they are degraded, this could explain why your antibiotic-susceptible E. coli can grow on your plates.
The second way of research is to make an antibiogram/identification of your ampicillin/kanamycin-resistant strains to verify that your strain is not contaminated by another one.
Regards
It could be that the competent cells are contaminated with transformed bacteria
Your glassware had traces of plasmid DNA from other cultures. While you are growing and treating the competent cells they can take in plasmids on the glassware very efficiently. Use dedicated glassware or at least acid wash them.
The two most logical answers are that either your media is bad (incorrect antibiotic concentration or antibiotic stock solutions have gone bad) or if these are home-made competent cells they were made from the wrong strain.
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