To insert a fragment of DNA that is to be cloned into a cloning vehicle.
A cloning vector must be available in sufficient quantity to use. A source of DNA also must be available.
Ligation precedes transformation. This is the process through which a piece of DNA is inserted into a vector such as a plasmid. Many strategies and protocols are available. The effect of incubating a vector and "insert," first with a restriction enzyme, and then with some ligating or other similar enzyme, is illustrated in the movie. The cleavage site on the plasmid is often located amidst a particular gene (e.g., β-galactosidase). Subsequently, only those colonies in which the function of the gene has been inhibited would be selected for further study. Several clever strategies for performing insertions amidst genes in the vector have been developed which offer possible techniques for selecting suitable transformants. The digested vector often is treated with a phosphatase (BAP or CIP). This removes the phosphate groups from the ends of the vector and prevents re-closure of the vector and thus increases ligation efficiency.
Ligation consists of mixing and incubating materials. The usual components include: a mixture of salts, ATP, and dithiothreitol or β-mercaptoethanol, etc.; T4 DNA ligase; cloning vehicle, such as a plasmid or phage; the passenger DNA, perhaps from electroelution; and buffer. Keep the materials on ice; several components are unstable at room temperature. Incubate the mixture at room temperature for 2-4 hours, or at 15oC for 15 hours, or up to 20 hours at 4oC.
An aliquot of this mixture can be used immediately for transformation (A-22) or stored frozen at -20oC for transformation at a later time.