Hello. I'm writing with courage to seek advice from professors and researchers. Currently, I am researching the impact of introducing a B plasmid into E. coli carrying an A plasmid, to study the effect of B genes on A genes.
After transforming the A plasmid (ampicillin resistance) via electroporation, I culture the cells to create electroporation competent cells, and then perform electroporation transformation with the B plasmid (gentamicin resistance).
While transforming A and B separately into DH10B Competent cells works well, colony formation does not occur when transforming B into A-harboring competent cells.
A plasmid: ColE1 origin, B plasmid: oriV, so there should be no incompatibility issues.
I wonder if adding ampicillin (1X, 100mg/ml) during the culturing process after transforming A could affect the cells. I tried dividing the cultures into small cultures, always adding 1x ampicillin, and when doing large cultures, I tried not adding antibiotics, or adding them at 0.2x concentration, but in all cases, transformation hardly occurs.
Should I consider anything else? Could co-transformation be the solution?
I would greatly appreciate your help. Please let me know if you need more information for your response.
It is hard to know what the problem is, could the genes you have cloned on the plasmids somehow be incompatible?
But you could try to reverse the order (do plasmid B first).
Secondly co-transformation usually works fine, however what you are doing should also work. So if there is some compatibility problem then it would occur regardless.
As a control you could try to transform the plasmid B parent plasmid into your competent cells.
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