AbstractThe reaction conditions and applications of two phosphatases and one kinase are described in this unit. Bacterial alkaline phosphatase (BAP) from E. coli and calf intestine phosphatase (CIP) from veal are commonly used in nucleic acid research. Both enzymes catalyze the hydrolysis of 5′‐phosphate residues from DNA, RNA, and ribo‐ and deoxyribonucleoside triphosphates. The dephosphorylated products possess 5′‐hydroxyl termini which can subsequently be radioactively labeled using [γ‐32P]ATP and T4 polynucleotide kinase. T4 polynucleotide kinase has 3 activities: the forward reaction efficiently catalyzes the transfer of the terminal (γ) phosphate of ATP to the 5′‐hydroxyl termini of DNA and RNA. The exchange reaction catalyzes the exchange of 5'‐terminal phosphates. Lastly, T4 polynucleotide kinase is a 3′ phosphatase.