Library construction in Pl phage vectors

Abstract The Pl cloning system was developed as an alternative to YAC and cosmid technology for the cloning of high molecular weight DNA fragments. It permits the recovery of inserts in E. coli that are up to 95 kb in size (more than twice the size of inserts that can be recovered in cosmids) with an efficiency that is intermediate between ;... cosmid and YAC cloning; greater than 105 clones can be generated using the Pl system with 1-2 µg of vector arms and 2-4 µg of Sau3Al digested, size selected genomic DNA insert (1). While the size of cloned insert recovered with the Pl system are significantly smaller than arc those recovered with YACs, the former offen, several advantages: libraries representative of multiple copies of the genome arc more easily produced, larger quantities of a particular cloned DNA are more easily isolated, the cloning process may be more faithful (2, 3), and physical dissection of cloned inserts may be more easily accomplished. Indeed, the ability to analyse cloned DNA before and after attempting amplification with the Pilytic replicon permits one to assess whether amplification of the insert by a high copy number replicon can indeed occur, and if so, whether DNA integrity is maintained.