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. Competing Interest Statement The authors have declared no competing interest. Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.