pELMO, an optimised in-house cloning vector

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• 作者：Andrea E. Ramos ; Marina Muñoz ; Darwin A. Moreno-Pérez ; Manuel A. Patarroyo
• 关键词：Recombinant DNA technology ; Cloning vector ; ccdB killer gene ; Blunt ; ended ; PCR cloning
• 刊名：AMB Express
• 出版年：2017
• 出版时间：December 2017
• 年：2017
• 卷：7
• 期：1
• 全文大小：1517KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Microbiology; Microbial Genetics and Genomics; Biotechnology;
• 出版者：Springer Berlin Heidelberg
• ISSN：2191-0855
• 卷排序：7

文摘

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 SmaI 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.