High-throughput cloning for proteomics research.
Ligation-independent cloning (LIC) is a simple, rapid, and efficient method for high-throughput cloning. In this system, linear plasmid vector and insert DNA are treated to generate complementary single-stranded overhangs that anneal during a short incubation. The LIC system is adaptable for use with any vector following an alteration of the vector sequence. This chapter describes the creation of an LIC-compatible vector, with tips on how to make any vector LIC-enabled. It also includes a protocol for generating high-quality linearized vector template for the LIC reaction. Lastly, a step-by-step protocol of the LIC reaction is outlined, with useful tips and tricks for optimization and screening.
Doyle
Sharon A
SA
Proteomics Group, DOE Joint Genome Institute, Walnut Creek, CA, USA.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D040901:Proteomics / Q000379:methods*
D018076:DNA, Complementary