Cell survival and genome rescue after UV irradiation in Escherichia coli depends on DNA repair mechanisms induced in response to DNA damage as part of the SOS regulon. SOS occurs in two phases. The first phase is dominated by accurate repair processes such as excision and recombinational DNA repair, while the second phase is characterized by a large approximately 100-fold increase in mutations caused by an error-prone replication of damaged DNA templates. SOS mutagenesis occurs as a direct result of the action of the UmuDC gene-products, which form the low fidelity Escherichia coli DNA polymerase V, a heterotrimeric complex composed of UmuD'(2)C. This chapter describes the preparation of highly purified native pol V that is suitable for a wide range of biochemical studies of protein-protein, protein-DNA interactions and translesion-synthesis (TLS) mechanisms.