pIDDLE6: A system for ligation and expression-cloning in E. coli

Summary The pIDDLE6 (for PCR-based, in-frame, directional DNA ligation and expression) system utilises novel ligation procedures to clone and overexpress almost any coding sequence. Inserts are cloned either by blunt ligation in the presence of PmeI or by ligation between dissimilar SfiI sites. A chimeric protein with both N- and C-terminal tags is produced, allowing purification by via starch-or nickel-affinity under native or denaturing conditions. The tags can be removed independently. The the vector and ligation procedures have been used successfully as described.