FAQ: Can PCR products be phosphorylated in the PCR mixture?

No, because the ammonium sulfate found in most PCR buffers inhibits T4 Polynucleotide Kinase (NEB #M0201). Instead, gel purify your DNA or use a G25 spin column. In a 50 µl reaction of 1X Kinase Reaction Buffer use up to 200 pmol of 5´-hydroxyl DNA termini with 1 mM ATP. Heat to 70°C for 5 minutes, then chill on ice. Add 20 units T4 Polynucleotide Kinase (NEB #M0201) and incubate at 37°C for 30 minutes. Alternatively, treat your primers with T4 Polynucleotide Kinase (NEB #M0201) prior to PCR amplification.