Hi,
I am doing transformation experiments with plasmidic extract and I am using DH5a cells for the transformation..I obtained few colonies on a plate with 1 ug/ml of meropenem antibiotic. Anyway, they are not transformant because I have done PCR for the genes carried by the plasmids (and plasmidic extraction as well) and I did not obtain anything. Is it possible that the colonies I have are simply the strain that acquired some sort of resistance at low concentration of the antibiotic?What would be the cause for this? Some mutation?
Thanks
Perform a negative control transformation with no plasmid DNA. This will give you an idea of the rate of spontaneous resistance.
It may also be possible that your PCR reaction did not work.. sometimes plasmid purrification kits dont work properly
A possible cause would be a contamination with a resistant E. coli DH5alpha where you perform your transformations experiments, that is not infrecuent if you have been working with this antibiotic in the place for some time. I recommend to prepare new solutions and take additional precautions in order to sterilize the work zone.
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