A series of protocols are presented for the storage, growth, transformation, and characterization of wild type and mutant strains of Synechocystis sp. strain PCC 6803. These protocols include the isolation of genomic DNA and the strategies required for the construction of specific gene interruptions or deletions in this organism. This cyanobacterium has been used widely as a model for photosynthesis research, and the sequence of its genome is available at CyanoBase (http://genome.kazusa.or.jp/cyanobase/). The details provided in this chapter do not assume any previous experience in working with cyanobacteria and are intended to enable new investigators to take advantage of a wide range of gene modification and mutation mapping techniques that have been adapted for use in this system.