Hello colleagues,
I'm trying to transfer the plasmid pKEB31:GOI carried by E. coli DH5alfa on Pantoea agglomerans using E. coli pRK2073 as helper. I did this triparental mating lots of time with different plasmid but all smaller than pKE31 which is 14.5 kb.
After the mating I plate different dilutions of bacterial mixture on plates enriched with Pantoea and pKE31 antibiotics and incubate them overnight at 28C (optimun for Pantoea).
The day after my plate were empty.
What could have gone wrong?
It might be possible the plasmid is to big?
Hello, 14.5bp is not so big for triparental mating. May be something wrong with procedure.
There really is no size limit, you should be able to transfer plasmids that are hundreds of kb during a mating. So 14kb should not be a problem at all. I would double check all your strains and plasmids. Are you certain that ccdB was deleted when your GOI was cloned into pKEB31? Because the wild type plasmid will not survive in DH5alpha.
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