An artifact of recombinatorial cloning challenges established beliefs of plasmid cotransformation, selection, and...

Gateway cloning is an easy, efficient, accurate, and versatile cloning strategy. During Expression clone validation, we sometimes see an additional band co-migrating with the pDONR (Entry) backbone. We show that this "mystery" band is not an artifact of aberrant recombination but instead originates from a cotransformation event, where more than one different plasmid is transformed into a single Escherichia coli cell simultaneously and in the absence of antibiotic selection. We find that the unselected pDONR Entry plasmid is cotransformed into E. coli with the desired Expression vector in 9%-29% of colonies and is maintained without antibiotic selection, despite plasmid incompatibility. We propose an easy strategy to screen for and eliminate cotransformants. Our results challenge accepted beliefs of bacterial plasmid transformation, selection, and maintenance and comprise the first documented occurrence of cotransformation in Gateway cloning.