A protocol for ligation-dependent cloning using the Flexi Vector method in a 96-well format is described. The complete protocol includes PCR amplification of the desired gene to append Flexi Vector cloning sequences, restriction digestion of the PCR products, ligation of the digested PCR products into a similarly digested acceptor vector, transformation and growth of host cells, analysis of the transformed clones, and storage of a sequence-verified clone. The protocol also includes transfer of the sequence-verified clones into another Flexi Vector plasmid backbone. Smaller numbers of cloning reactions can be undertaken by appropriate scaling of the indicated reaction volumes.