Method for directional cloning

A method for directional cloning, the method comprising:

• providing a target vector and an insert Deoxyribonucleic acid (DNA) segment;
• and
• mixing the target vector, the insert DNA segment, an amount of a restriction enzyme, and an amount of a DNA ligase in a container to form a mixture;
• incubating the mixture at a predetermined temperature about 37° C for about 10 minutes to:
• cleave the target vector with the restriction enzyme at the predetermined temperature, and
• ligate at least one portion of the insert DNA segment to the cleaved target vector at the predetermined temperature to generate a recombinant DNA molecule comprising at least one portion of the target vector and the at least one portion of the insert DNA segment;
• and
• mixing a portion of the mixture with competent host cells about 10 minutes after the mixing the target vector, the insert DNA segment, the amount of the restriction enzyme, and the amount of the DNA ligase in the container to transform the generated recombinant DNA molecule into the competent host cells, wherein the insert DNA segment is a doube-stranded linearized polynucleotide and the target vector is a closed circular plasmid before the incubating the mixture at the predetermined temperature about 37° C. for about 10 minutes.