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Journal of Structural and Functional Genomics

, Volume 7, Issue 3–4, pp 109–118

First Online: 13 February 2007Received: 21 August 2006Accepted: 14 December 2006

Abstract

Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput HTP cloning method. We implemented a modified Enzyme Free Cloning EFC procedure, a PCR-only method that eliminates all variables other than PCR efficiency by circumventing enzymatic treatments. We compared the cloning efficiency of EFC with that of Ligation Independent Cloning LIC. Both methods are well suited for HTP cloning, but EFC yields three times more transformants and a cloning efficiency of 91%, comparable with recombinational cloning methods and significantly better than LIC 79%. EFC requires only nanogram amounts of both vector and insert, does not require highly competent cells and is, in contrast to LIC, largely insensitive to variations in PCR product concentration. Automated protein expression screening of expression strains directly transformed with EFC reactions showed, that the traditional preceding step via a cloning strain can be circumvented. EFC proves an efficient and robust HTP cloning method, that is compatible with existing Ligation Independent Cloning vectors, and highly suitable for automation.

KeywordsLigation independent cloning Enzyme free cloning High throughput Functional genomics Recombinant protein expression PCR-only cloning  Download fulltext PDF



Autor: Rob N. de Jong - Mark A. Daniëls - Rob Kaptein - Gert E. Folkers

Fuente: https://link.springer.com/







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