T4 polynucleotide kinase (PNK) catalyzes the transfer of terminal phosphate from the gama position of rATP to the 5'-hydroxyl terminus of polynucleotides (double- and single-stranded DNA or RNA) and nucleoside 3'-monophosphates. PNK also catalyses the exchange of 5'-terminal phosphates and exhibits 3'-phosphatase activity.

Applications: End-labeling DNA or RNA, addition of 5’ -phosphates to oligonucleotides, removal of 3’ -phosphoryl groups

Storage buffer:

• 50 mM KCl

• 10 mM Tris-HCl (pH 7.8)

• 0.1 mM EDTA

• 1 mM DTT

• 0.1 μM ATP

• 50% glycerol

Reaction Buffer (10x):

• 100 mM Tris-HCl (pH 7.5)

• 50 mM MgCl₂

• 50 mM DTT

The efficiency of blunt and recessed 5’ -end phosphorylation can be improved by heating to 70°C for 5 minutes, then chilling on ice prior kinase addition.

Since polynucleotide kinase is inhibited by ammonium ions, DNA should not be precipitated in the presence of ammonium ions prior to phosphorylation.

Quality Assurance: Free of exonuclease, phosphatase, endonuclease and RNase activities

Unit Definition: One unit is the amount of enzyme catalyzing the transfer of 1 nmol of phosphate in 30 minutes at 37°C

Storage conditions: -20°C