Establishment Of Molecular Cloning Procedure Using A Novel Microbial Cell Extract

Abstract

In-fusion cloning is the molecular cloning techniques applying the principle of Ligation Independent Cloning (LIC) method. This technique requires the specific designed primers which is exonuclease-based cloning that utilizes the 3' to 5' exonuclease activity to construct single-stranded 5' overhangs and the complementary single standard overhangs anneal together. Thereby, its potential advantages include directional cloning with one or multiple DNA fragments cloned into any vector, cloning any insert, into any position, within any vector. Currently, In-fusion cloning kit is commercialized by several biotechnology cooperations (e.g Takarabio), but the cost per ligation reaction of current commercial kits is quite high. This study has found out the optimal ligation parameters (i.e plasmid to insert ratio, ligation duration, and ligation temperature) using novel microbial cell extract [Eclone, gifted by Laboratory of molecular biotechnology, University of Science], which is being developed based on the mechanism of In-fusion cloning method. The outcome of this thesis will be a standardized protocol for molecular cloning using a novel enzyme to reduce the cost and materials for each ligation reaction. In the scope of this study, the molecular cloning procedure using a novel microbial cell extract was established with the optimal parameter in ligation process at 25°C, the ratio of insert to plasmid [1:1] within 15 minutes. This condition resulted in high efficiency, less ligation time, and lower cost per ligation reaction.