PNK Treatment of DNA Ends

This step is used to add a phosphate group to the 5' end of a DNA molecule. Most primers for example are ordered without this being added as it requires an extra synthesis step and hence greater cost. However subsequent ligation steps are more efficient if these phosphate groups are added.

• T4 Polynucleotide Kinase is an enzyme that can perform this on blunt or overhanging DNA ends.

• They describe a protocol for use that is modified and summarized below.

• Reaction Mix
  • 1[math]\displaystyle{ \mu }[/math]l PNK stock (10,000U/ml)
  • 1[math]\displaystyle{ \mu }[/math]l T4 Ligase Buffer
  • 8[math]\displaystyle{ \mu }[/math]l Substrate

• Reaction Conditions
• 37^oC for 30mins
• 65^oC for 20mins
• Store at 4^oC

• The T4 Ligase Buffer provides the required ATP and substitutes for the PNK Buffer and ATP in the NEB protocol. It is actually possible to perform the PNK step and a ligation step simultaneously although the author has not done this.