First, i PCR amplyfied my gene (~ 2500bp) and then cloned it to the bacteria (DH5@).
Second, i digested it with restriction enzymes (blunt end, ~1300bp) and left flanking sites (~1200bp) inside the vector.
Third, i cloned pGFPuv into the vector instead of 1300bp length gene. Finally i had recombinant bacteria but the flanking sites into the rec. bacteria was deleted.
I thought that it could be toxic for the bacteria and i cultured it at RT but nothing happened. It was still deleted.
Anybody has suggest?