Cloning in plasmid vectors.

Abstract

A fundamental step in molecular biology is the cloning of a DNA fragment insert into a plasmid vector. This allows the cloned fragment to be replicated upon transformation of the recombinant molecule into a bacterial cell (see Chapters 4 and 5) so that the DNA of interest can be investigated further. Cloning is an essential part of many experiments, including library generation (see Chapter 18) and expression studies (see Chapter 29). The vector and insert DNA are usually digested with a type II restriction endonuclease that cleaves at specific sites in the DNA. The two molecules must have compatible ends for cloning to proceed. The generation of compatible ends requires the use of restriction and modifying enzymes, which ultimately results in the generation of either blunt or overhanging ends. The cleaved fragments are mixed in the presence of DNA ligase that produces a mixture of products, some of which should consist of the vector containing the inserted DNA fragment. Numerous vectors are commercially available to facilitate cloning for various applications (see Chapter 2). Chapter 14 presents a detailed description for designing cloning experiments using such vectors. Additionally, specialized vectors are available for cloning polymerase chain reaction (PCR) products generated by Taq DNA polymerase (see Chapter 17). This chapter focuses on the preparation of DNA fragments for efficient cloning.