The DNA Fragmentation Kit is designed to perform random genomic DNA fragmentation and other long-chain dsDNA fragmentation via enzymatic treatment followed by blunting of the resulting DNA fragments. The kit does not require an additional physical shearing step using an apparatus such as a sonicator. Blunt-end DNA fragments may be ligated with blunt-end vectors. If blunting is not required, the reaction may be stopped after DNA fragmentation is complete. The kit may also be used to pretreat samples enriched with methylated DNA and to prepare DNA samples for sequencing.