Attaching Adaptors to Protruding Termini Protocol Adaptors are short double-stranded synthetic oligonucleotides that carry an internal restriction endonuclease recognition site and single-stranded tails at one or both ends. Adaptors are used to exchange restriction sites at the termini of linear DNA molecules. They may be purchased in phosphorylated and unphosphorylated forms.
Fragmentation of DNA by Nebulization Protocol Protocol describes a method for DNA fragmentation by nebulization, in which the fine mist created by forcing a DNA solution through a small hole in the nebulizer unit is collected. The size of the fragments obtained by nebulization is determined chiefly by the speed at which the DNA solution passes through the hole, altering the pressure of the gas blowing through the nebulizer, the viscosity of the solution, and the temperature.
Fragmentation of DNA by Sonication Protocol Protocol describes a method for DNA fragmentation by sonication. During sonication, DNA samples are subjected to hydrodynamic shearing by exposure to brief periods of sonication. DNA that has been sonicated for excessive periods of time is extremely difficult to clone.
Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose Protocol Protocol for ligating plasmid and target DNAs in low-melting-temperature agarose. Ligation in low-melting-temperature agarose is much less efficient than ligation with purified DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for rapid subcloning of segments of DNA in dephosphorylated vectors and assembling recombinant constructs.
Ligation Optimization Protocol Protocol can be used to optimize ligation conditions for difficult to clone (e.g. very large) fragments. The principle is to independently characterize the ligation kinetics of the vector and insert DNA fragments and then to combine them in optimal ratios.