The most likely cause of incomplete phosphorylation is oxidized DTT in the T4 Polynucleotide Kinase (PNK) reaction buffer (DTT oxidation occurs naturally and is acclerated by repeated freeze/thaw cycles or excessive heating). Use fresh buffer (< 1 year) or add fresh DTT to 5 mM using a 1M stock. Other factors include: a. An excess of salt, phosphate or ammonium ions present: PNK is inhibited by high levels of salt (50% inhibition by 150 mM NaCl), phosphate (50% inhibition by 7 mM phosphate) and ammonium ions (75% inhibited by 7 mM (NH4)2SO4). NEB's ThermoPol reaction buffer contains 10 mM (NH4)2SO4 which must be removed before performing a kinase reaction. DNA should not be precipitated in the presence of ammonium ions prior to phosphorylation. Drop dialyze or use a commercially available spin column to remove salt from the sample. b. Small nucleic acid contaminants in the DNA preparation: It is important that the substrate be purified by size selection to remove low molecular weight nucleic acid contaminants which can represent a high percentage of total 5´-OH ends, even if the total mass is low. Use a commercially available spin column to remove these contaminants as well as excess salt. c. The end is recessed or blunt: If the ends are blunt-ended or 5´-recessed, heat the substrate/buffer mixture for 10 minutes at 70°C, chill rapidly on ice before adding the ATP and enzyme, then incubate at 37°C. This helps the PNK access the 5´-OH termini. d. The phosphatase was not inactivated: To remove CIP or BAP use phenol/CHCl3. Antarctic Phosphatase and SAP can be heat inactivated. e. ATP not added: PNK requires ATP for activity. Typically, PNK reactions are followed by a ligation reaction. To simplify this process, PNK is optimized for use in T4 Ligase reaction buffer (which contains the appropriate amount of ATP). We recommend performing the PNK reaction in Ligase buffer for 30 minutes; you can then proceed to ligation without a buffer change or heat inactivation; however PNK will remain active in the subsequent reaction unless heat inactivated or removed (spin column, gel purification) prior to the next step.