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

    Protocol

    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.