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T4 Polynucleotide Kinase (PNK) catalyzes the transfer of the gamma-phosphate of ATP to the 5'-hydroxyl termini of DNA or RNA. This phosphate transfer is commonly referred to as a kinase or phosphorylation reaction ([gamma-32P]ATP is often used in the reaction). Nucleic acids with a 5'-hydroxyl (OH) group can be added directly to a kinase reaction.
Nucleic acids with a 5'-phosphate should be dephosphorylated (e.g., with Calf Intestinal Phosphatase) prior to labeling with PNK and [gamma-32P]ATP. Inactivating the Calf Intestinal Phosphatase (CIP) after the dephosphorylation reaction typically requires phenol extraction and ethanol precipitation, although the KinaseMaxØ 5' End Labeling Kit includes a novel Phosphatase Removal Reagent for quick and complete removal of CIP without phenol extraction or ethanol precipitation. After the dephosphorylation step, the reagent is added to the reaction and incubated at room temperature for 2 minutes. The tube is then spun for a minute in a microfuge and the supernatant transferred to a new tube for the kinase reaction.
The below protocol is for 5' end labeling RNA with PNK. The labeled RNA can be used as a probe or for RNA structure assessment, protein footprinting, and boundary determination experiments.