Ligation-independent cloning for plant research

Abstract

Molecular cloning is a vital step in much of today’s plant biological research. Particularly, when a species is amenable to transgenic manipulation, cloning enables detailed study of gene and protein function in vivo. Therefore, accurate, consistent, and effi cient cloning methods have the potential to accelerate biological research. Traditional restriction-enzyme/ligase-based strategies are often ineffi cient, while novel alternative methods can be less economical. We have recently optimized a method for Ligation-Independent Cloning (LIC) that is both effi cient and economical. We have developed a large set of LIC-compatible plasmids for application in plant research. These include dedicated vectors for gene expression analysis, protein localization studies, and protein misexpression. We describe a detailed protocol that allows the reliable generation of plant transformation-ready constructs from PCR fragments in 2–3 days.