Journal
BIOTECHNIQUES
Volume 63, Issue 3, Pages 125-130Publisher
BIOTECHNIQUES OFFICE
DOI: 10.2144/000114588
Keywords
seamless cloning; SLIC; T4 DNA polymerase
Funding
- Korea Institute of Ocean Science and Technology (KIOST) [PE99514]
- Ministry of Oceans and Fisheries in the Republic of Korea [PM60120]
- Korea Institute of Marine Science & Technology Promotion (KIMST) [PE99514] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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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 degrees C). The highest cloning efficiency resulted when inserts with homology lengths <20 bases were treated with T4 DNA polymerase for 30 s at 50 degrees 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.
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