Each ligation reaction generally contains 1-10 Weiss units of high quality ligase. The bacterial strain chosen for transformation of the ligation reaction and subsequent propagation of the recombinant plasmid may need to be optimized, as well as the temperature at which the bacteria are grown. The concentration of antibiotic present for selection of the recombinants may need to be optimized. This inhibits self-ligation and favors insert ligation. For successful ligation reactions, both the vector and insert DNA should be very clean.
Contaminants can interfere with the ligase and inhibit the reaction if present.
Systems may be utilized to purify both the vector and insert DNA prior to the ligation reaction. In order to be certain that a subcloning experiment has been successful, and to troubleshoot if the desired clone is not obtained, appropriate controls should be included with each experiment. In order to ensure that the host cells are competent and not contaminated, three tests are recommended. Equal volumes of these cells should be plated on medium with and without antibiotic selection. The selective plate should contain no colonies unless there is a contaminating plasmid or if the antibiotic is not efficacious.