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In-Situ Gel-Free Plasmid Reassembling for Rapid Gene Subcloning and Truncation
Jamal-Livani, Narges; Nikokar, Elham; Safdari, Yaghoub.
  • Jamal-Livani, Narges; Golestan University of Medical Sciences. School of advanced Technologies in Medicine. Department of Medical Biotechnology. Gorgan. IR
  • Nikokar, Elham; Golestan University of Medical Sciences. School of advanced Technologies in Medicine. Department of Medical Biotechnology. Gorgan. IR
  • Safdari, Yaghoub; Golestan University of Medical Sciences. Golestan Research Center of Gastroenterology and Hepatology. Gorgan. IR
Braz. arch. biol. technol ; 63: e20190223, 2020. tab, graf
Article in English | LILACS | ID: biblio-1132217
ABSTRACT
Abstract Gene subcloning, a process in which the nucleotide sequence of interest is excised from on plasmid and inserted into another, seems to be an easy task to done. However, not all subcloning attempts are successful, even when the insert sequence and the double digested target plasmid are successfully purified form agarose gel and thought to be ready for subsequent ligation. In the current study we introduce a reliable, easy, and time consuming method for gene subcloning and also truncation. The stages are all carried out in a single microtube without any running on a gel, making it possible to accomplish a successful gene subcloning or truncation even with low concentrations of DNA molecules. Summarily, subcloning is achieved by mixing the plasmids of interest in a microtube and subjecting to restriction enzymes whose restriction sites flank the segment that is going to be subcloned. Digestion mixture is precipitated in the same microtube using isopropanol and the resultant DNA molecules are allowed to take part in a ligation reaction. The recombinant plasmids of interest are screened by colony PCR. Truncation is achieved by double- digestion of the plasmid of interest using a restriction enzyme whose restriction site flanks the segment that is going to be cut out.
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Full text: Available Index: LILACS (Americas) Main subject: Plasmids / Cloning, Molecular / Genetic Vectors Language: English Journal: Braz. arch. biol. technol Journal subject: Biology Year: 2020 Type: Article Affiliation country: Iran Institution/Affiliation country: Golestan University of Medical Sciences/IR

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Full text: Available Index: LILACS (Americas) Main subject: Plasmids / Cloning, Molecular / Genetic Vectors Language: English Journal: Braz. arch. biol. technol Journal subject: Biology Year: 2020 Type: Article Affiliation country: Iran Institution/Affiliation country: Golestan University of Medical Sciences/IR