![]() Received: FebruAccepted: MaPublished: April 13, 2016Ĭopyright: © 2016 Benoit et al. Lustig, Tulane University Health Sciences Center, UNITED STATES ![]() We successfully used this modified Gibson assembly protocol with two short insert-plasmid overlap regions, each counting only 15 nucleotides.Ĭitation: Benoit RM, Ostermeier C, Geiser M, Li JSZ, Widmer H, Auer M (2016) Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost. These findings demonstrate that highly efficient insert-plasmid assembly can be achieved by using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, which significantly reduces the cost of the reactions. Ligation of the remaining nicks did not lead to a further increase in transformation efficiency. Accordingly, filling-in of single-stranded gaps using DNA polymerase resulted in increased transformation efficiency. Most importantly, our data show that single-stranded gaps in double-stranded plasmids, which occur in typical SLIC protocols, can drastically decrease the efficiency at which the DNA transforms competent E. ![]() With the aim to improve the robustness of seamless cloning experiments while keeping costs low, we examined the importance of complementary single-stranded DNA ends for co-transformation cloning and the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids.
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