T4 Polynucleotide Kinase

T4 Polynucleotide Kinase catalyzes the transfer and exchange of Pi from the γ position of ATP to the 5´ -hydroxyl terminus of polynucleotides (double-and single-stranded DNA and RNA) and nucleoside 3´-monophosphates. T4 Polynucleotide Kinase also catalyzes the removal of 3´-phosphoryl groups from 3´-phosphoryl polynucleotides, deoxynucleoside 3´-monophosphates and deoxynucleoside 3´-diphosphates.

The enzyme is available in 500 and 2500 unit sizes at a concentration of 10 U/µL. The enzyme is supplied with a 10× Reaction Buffer.

Applications

• End-labeling DNA or RNA for probes and DNA sequencing.
• Addition of 5´-phosphates to oligonucleotides to allow subsequent ligation.
• Removal of 3´-phosphoryl groups.

Product Specifications

• Storage Buffer: 10 mM Tris-HCl (pH 7.4 at 25 ^oC), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.1 µM ATP, and 50% (v/v) glycerol.
• Unit Definition: One unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmol of acid-insoluble [³²P] in a total reaction volume of 50 μl in 30 minutes at 37°C in 1X Reaction Buffer with 66 µM [γ-³²P] ATP (5 x 10⁶ cpm/µmol) and 0.26 mM 5´-hydroxyl-terminated salmon sperm DNA.

Phosphorylation Protocol with T4 PNK

1.     Assemble the following reaction in a microcentrifuge tube on ice:

        10× Reaction Buffer                  5 µL
        10 mM ATP                               5 µL
         DNA                                          up to 300 pmol of 5’ termini
         Nuclease-free water                  to 49 µL
        T4 PNK                                     1 µL
        Total volume                             50 µL

2.     Mix gently and spin down for a few seconds.

3.     Incubate at 37 °C for 30 min.

4.     Stop the reaction by heating at 65 °C for 20 min.

End-labeling Protocol with T4 PNK

1.  Assemble the following reaction in a microcentrifuge tube on ice:

       10× Reaction Buffer                                 2 µL
        ³²P ATP (3,000 Ci/mmol, 5 mCi/ml)         1 µL
       DNA                                                         1 µg
       Nuclease-free water                                 to 19 µL
       T4 PNK                                                    1 µL
       Total volume                                            20 µL

2.  Mix gently and spin down for a few seconds.

3.  Incubate at 37 °C for 30 min.

4.  Run the samples for 50 to 60 minutes at 100V in TBE buffer in a 4-20% acrylamide gel (10 cm x 10 cm). A 20 minutes exposure gives very readable signals.