Patent ID: 9567593
Date: 2017-02-14
CPC Classifications: C12N

Claim:
1. A method for directional cloning, the method comprising the following steps in the order named: (1) providing a closed circular plasmid, a double-stranded linearized polynucleotide, an amount of a restriction enzyme, and an amount of a DNA ligase; (2) incubating the closed circular plasmid, the double-stranded linearized polynucleotide, the amount of the restriction enzyme, and the amount of the DNA ligase in a single container at about 37° C. for about 10 minutes, thereby cleaving the closed circular plasmid with the restriction enzyme, ligating at least one portion of the double-stranded linearized polynucleotide to the cleaved closed circular plasmid to generate a recombinant DNA molecule comprising at least one portion of the closed circular plasmid and at least one portion of the double-stranded linearized polynucleotide, thus obtaining a mixture comprising the recombinant DNA molecule; and (3) mixing a portion of the mixture with competent host cells about 10 minutes after starting the incubation of step (2), wherein: the restriction enzyme is at a concentration of about 20 units in a volume of 20 μl of the mixture, the restriction enzyme comprises at least one of BsaI, BbsI, BsmBI, Alw26I or LguI, and the DNA ligase is at a concentration of about 5 units in a volume of 20 μl of the mixture.