I am trying to insert in a plasmid the ORF of a gene I am interested in my project. This plasmid is able to conditionally express the inserted sequences under the control of a b-actin promoter. Between the promoter and its downstream sequence, indeed, it's present a stop codon flanked by to flox sequences. So the expression of a given sequence inserted downstream of the promoter is activated only in presence of Cre-recombinase. I will use this construct to activate my gene of interest in a transgenic mouse expressing the Cre only in some territories of the brain. My problem is that when I try the ligation and electroporate it in normal bacterial strains, such as XL1blue, topten, etc. i observed a certain rate of recombination. The inserted sequence, or even the plasmidic sequence are shortened or inverted. I suspect a recombination event. I am not an expert in this field, but I know that there are bacterial strains with a lower rate of recombination. Could you suggest me such bacterial strains or viable protocols to overcome these problems?
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Gene Expression