5’-Phosphorylation of Oligonucleotides prior to using Phusion Site-Directed Mutagenesis Kit (F-541)


  1. Add the following components to a microcentrifuge tube:
    300 pmol oligonucleotide
    5 µl 10X T4 Polynucleotide Kinase Reaction Buffer
    1 µl T4 Polynucleotide Kinase 10 U/µl (NEB# M0201S/L )
    5 µl 10 mM ATP
    H2O to a final volume of 50 µl
  2. Incubate the reaction at 37°C for 30 minutes.
  3. Inactivate the T4 Polynucleotide Kinase at 65°C for 20 minutes. Alternatively PNK reaction can be phenol-extracted and ethanol precipitated or purified with a commercial kit.
  4. The reaction products can be stored at -20°C or added directly to the mutagenesis reaction.