ligation independent cloning serial cloner

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