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Troubleshooting Guide for Phosphorylation

Problem Cause Solution
Few or no transformants
 Inefficient phosphorylation
  • Purify the DNA prior to phosphorylation. Excess salt, phosphate or ammonium ions may inhibit the kinase.
  • If the ends are blunt or 5´ recessed, heat the substrate/buffer mixture for 10 minutes at 70°C. Rapidly chill on ice before adding the ATP and enzyme, then incubate at 37°C.
  • ATP was not added. Supplement the reaction with 1mM ATP, as it is required by T4 Polynucleotide Kinase (NEB #M0201).
  • Alternatively, use 1X T4 DNA Ligase Buffer (contains 1 mM ATP) instead of the 1X T4 PNK Buffer