I am not so clear about what you said here: "They both received a quickchange product -introducing the same mutation in the WT form of the protein and a mutated form". Did you mean you transform two genes into the competent cells, one is the wild-type gene and the other one is a mutated form of the gene?

If so, probably the protein produced from the mutated gene cause that?! Can this gene express in E. coli?

Dear Yuan-Yeu,

I had 2 independent QC reactions, but both introduced the same substitution either in the WT protein or either in an already mutated form of the WT (another substitution somewhere else). It means the 2 vectors are identical except one bp... Is that a bit clearer now?

But anyway, I cannot imagine any effect related to protein expression (leaking) mostly because I use a double induction system (cold and IPTG) and how can I explain that 5 clones on one dish got similar "mutation/change" to induce protein expression.... And TOP10 is not an expression strain too.

I'm more thinking about a chemical/contaminant in these bunch of culture tubes that affect bacteria behaviour... because tubes are really what makes the culture unique (media, antibiotics all share a common source)... I read stress can induce aggregation in some strains...

Thanks,

Guillaume

Dear Ali,

thanks for your input. I see. Anyway, I got my vector amplified, yield is not an issue and sequencing is OK.

Best wishes,

Guillaume