Schematic diagram illustrating the cloning process that lead to detection of an extra DNA element integrated into the insert during the process. A PCR fragment, amplified from the gene of interest, with a size of about 4.5 kb, was inserted into a plasmid. The recombinant plasmid was then transformed into E. coli. The PCR was performed using the same sets of primers and extracted recombinant plasmids as a template. The resulting PCR fragment appeared to be about 5.8 kb long. This indicates the presence of extra DNA inside the insert. Further multiple restriction digestion analyses and sequencing confirmed the presence of the extra 1.3 kb DNA fragment within the insert.