pELMO, an optimised in-house cloning vector

• Published in: AMB Express Vol. 7; no. 1; p. 26
• Main Authors: Ramos, Andrea E., Muñoz, Marina, Moreno-Pérez, Darwin A., Patarroyo, Manuel A.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.12.2017; Springer Nature B.V
• Subjects: Biomedical and Life Sciences; Biotechnology; Life Sciences; Microbial Genetics and Genomics; Microbiology; Original; Original Article; Recombinant DNA technology; PCR cloning; Cloning vector; Blunt-ended; killer gene; ccdB killer gene
• ISSN: 2191-0855; 2191-0855
• DOI: 10.1186/s13568-017-0324-2

DNA cloning is an essential tool regarding DNA recombinant technology as it allows the replication of foreign DNA fragments within a cell. pELMO was here constructed as an in-house cloning vector for rapid and low-cost PCR product propagation; it is an optimally designed vector containing the ccdB killer gene from the pDONR 221 plasmid, cloned into the pUC18 vector’s multiple cloning site (Thermo Scientific). The ccdB killer gene has a cleavage site (CCC/GGG) for the Sma I restriction enzyme which is used for vector linearisation and cloning blunt-ended products. pELMO transformation efficiency was evaluated with different sized inserts and its cloning efficiency was compared to that of the pGEM-T Easy commercial vector. The highest pELMO transformation efficiency was observed for ~500 bp DNA fragments; pELMO vector had higher cloning efficiency for all insert sizes tested. In-house and commercial vector cloned insert reads after sequencing were similar thus highlighting that sequencing primers were designed and localised appropriately. pELMO is thus proposed as a practical alternative for in-house cloning of PCR products in molecular biology laboratories.