Successful completion of a semi-automated enzyme-free cloning method

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• 作者：Stefano Bonacci ; Scilla Buccato…
• 关键词：Laboratory automation ; Integrated robotic workstation ; High ; throughput ; Genomics ; Mutagenesis ; Cloning
• 刊名：Journal of Structural and Functional Genomics
• 出版年：2016
• 出版时间：September 2016
• 年：2016
• 卷：17
• 期：2-3
• 页码：57-66
• 全文大小：1,294 KB
• 刊物主题：Biochemistry, general; Animal Genetics and Genomics; Plant Genetics & Genomics; Microbial Genetics and Genomics; Bioinformatics; Human Genetics;
• 出版者：Springer Netherlands
• ISSN：1570-0267
• 卷排序：17

文摘

Nowadays, in scientific fields such as Structural Biology or Vaccinology, there is an increasing need of fast, effective and reproducible gene cloning and expression processes. Consequently, the implementation of robotic platforms enabling the automation of protocols is becoming a pressing demand. The main goal of our study was to set up a robotic platform devoted to the high-throughput automation of the polymerase incomplete primer extension cloning method, and to evaluate its efficiency compared to that achieved manually, by selecting a set of bacterial genes that were processed either in the automated platform (330) or manually (94). Here we show that we successfully set up a platform able to complete, with high efficiency, a wide range of molecular biology and biochemical steps. 329 gene targets (99 %) were effectively amplified using the automated procedure and 286 (87 %) of these PCR products were successfully cloned in expression vectors, with cloning success rates being higher for the automated protocols respect to the manual procedure (93.6 and 74.5 %, respectively).