An artifact of recombinatorial cloning challenges established beliefs of plasmid co-transformation, selection, and maintenance

Abstract

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 backbone. We show that this "mystery" band is not an artifact of aberrant recombination but instead originates from a co-transformation event. We find that the unselected pDONR plasmid is co-transformed 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 co-transformants. Our results challenge accepted beliefs of bacterial plasmid transformation, selection, and maintenance.