The Deletion Kit for Kilo-Sequencing is designed to facilitate DNA sequencing, especially sequencing 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). This kit can be used to create nested, unidirectional deletions in target DNA, enabling progressive movement of the vector's primer binding site towards the sequence of interest. The optimized reagents in the Deletion Kit for Kilo-Sequencing ensure efficient self-circularization of deletion subclones.