Efficient high-throughput gene cloning represents a critical first step for conducting functional and structural proteomics in the post-genomic era. The ligation-independent cloning (LIC) method has been almost universally adopted by large structural biology centers as a component of high-throughput structure determination pipelines. The LIC platform is easy to use, of low cost, and rapid, and importantly, it is easily adapted to 96- or 384-well format, thereby facilitating automation. Procedures are described for 96-well format cloning using the LIC technology.