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Biotechniques. 2017 Sep 1;63(3):125-130. doi: 10.2144/000114588.

Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning.

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Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Ansan, Republic of Korea.
Department of Marine Biotechnology, Korea University of Science and Technology, Daejeon, Republic of Korea.
Department of Biotechnology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh.


Previously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its cloning efficiency is occasionally poor, potentially due to formation of stable secondary structures within the single-stranded DNA (ssDNA) region generated by T4 DNA polymerase during the 2.5 min treatment at room temperature. To overcome this problem, we developed a modified thermo-regulated one-step SLIC approach by testing shorter T4 DNA polymerase treatment durations (5 s-2.5 min) over a wide range of temperatures (25-75°C). The highest cloning efficiency resulted when inserts with homology lengths <20 bases were treated with T4 DNA polymerase for 30 s at 50°C. This briefer T4 polymerase treatment at a higher temperature helps increase cloning efficiency for inserts with strong secondary structures at their ends, increasing the utility of one-step SLIC for the cloning of short fragments.


SLIC; T4 DNA polymerase; seamless cloning

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