ligation independent cloning serial cloner

SLIC uses a brief treatment of purified PCR products with the 3′→5′ exonuclease activity of T4 DNA polymerase to generate a higher proportion of recessed ends. PIPE relies on the observation that a significant portion of PCR products are incomplete, having 3′-recessed ends, particularly in the absence of a final extension step. The 3′ ends of the PCR primers are template-specific, whilst the 5′ ends incorporate tails specific for the cloning junction. (C) Colonies from ampicillin plates were patched onto kanamycin/X-gal plates to distinguish recombinants from unwanted insert vector or empty pUC18/Kan background colonies.Īll three techniques amplify the gene of interest by polymerase chain reaction (PCR). (B) Design of the reporter vector, encoding resistance for ampicillin and kanamycin, and the alpha-fragment of beta-galactosidase. For all techniques, a DpnI digestion step is included to remove plasmid template (not shown). In OEC, the insert PCR product acts a megaprimer to generate a nicked plasmid by overlap extension in vitro in a second round of amplification. In both these techniques, by amplifying vector and insert with primers containing complementary 5′-tails and mixing the products, the overhangs can anneal and are joined in vivo after transformation into E. In SLIC, purified PCR products are treated with T4 DNA polymerase (DNAP) so that the exonuclease activity will increase the proportion of recessed ends. (A) In PIPE, incomplete extension during PCR generates 3′-recessed ends. Principles of polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC) and overlap extension cloning (OEC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist.įigure 1. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: This work was supported by the National Health and Medical Research Council (1008081, and the National Heart Foundation of Australia (G11S5757, ). Received: OctoAccepted: NovemPublished: December 23, 2013Ĭopyright: © 2013 Stevenson et al.

PLoS ONE 8(12):Įditor: Jean Peccoud, Virginia Tech, United States of America These ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit.Ĭitation: Stevenson J, Krycer JR, Phan L, Brown AJ (2013) A Practical Comparison of Ligation-Independent Cloning Techniques. Our data suggest that for small inserts (<1.5 kb), OEC is a good option, requiring only two new primers, but performs poorly for larger inserts. Overall, PIPE achieved cloning efficiencies of ∼95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Here, we outline and optimise these techniques and identify important factors to guide cloning project design, including avoiding PCR artefacts such as primer-dimers and vector plasmid background. These strategies rely on the generation of complementary overhangs by DNA polymerase, without requiring specific restriction sites or ligation, and achieve high efficiencies in a fraction of the time at low cost. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), and overlap extension cloning (OEC). Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. The precise assembly of specific DNA sequences is a critical technique in molecular biology.

Ligation independent cloning serial cloner