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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.

Author information

1
Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Ansan, Republic of Korea.
2
Department of Marine Biotechnology, Korea University of Science and Technology, Daejeon, Republic of Korea.
3
Department of Biotechnology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh.

Abstract

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.

KEYWORDS:

SLIC; T4 DNA polymerase; seamless cloning

PMID:
28911316
DOI:
10.2144/000114588
[Indexed for MEDLINE]
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