Good morning,
We are trying to select our Agrobacterium tumefaciens colony with X-gal and IPTG assay since our plasmid has LacZalpha promoter. We cannot select the colonies by antibiotic selection since our bacteria is resistant to the antibiotic we have in our plasmid. Do you know if it can work in Agrobacterium? Which conditions are the best in order to see white and blue colonies? Thanks in advance.
Andrés Castillo , I think you have a very big problem due to having the same antibiotic resistanve gene in both plasmid and bacteria. In this case , you can not perform blue white selection because even a colony doesn’t contain plasmid, will grow in lb agar and this phenotype is normally belongs to vector inserted plasmid containing bacteria colony. At this point you will have a problem about selection of plasmid containing and B-gal gene disturbed colony. You must redesign your plasmid according to the bacteria‘s antibiotic resistance genes . Good luck.
Also there is another idea to select plasmid containing colony. You can perform colony PCR. After X-gal treatment, you will eliminate the cells do not contain your interest gene, than random selection of colonies for colony pcr may help you to find plasmid containing colony.
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