The Deletion Kit for Kilo-Sequencing is designed to aid sequence analysis of long DNA fragments cloned into the multiple cloning sites of M13 phage vectors (mp18/19) or pUC-related plasmid or phagemid vectors (pUC18/19). The kit is used to create nested unidirectional deletions in the target DNA, thereby progressively moving the primer binding site on the vector closer to the sequence of interest. Optimized reagent cocktails are included to ensure efficient self-circularization of the deletion subclones.