Phosphorylation of PCR Products with T4 Polynucleotide Kinase

Gregory Broussard

Revision: 1/25/11

(Please use, change and disseminate freely!)

Reaction:

3.5µL            diH2O

40µL            PCR Product

5µL            T4 PNK Buffer (10X)  (note 1 – use T4 DNA ligase buffer instead)

0.5µL            ATP (100 mM)  (note 1 – use T4 DNA ligase buffer instead)

1µL            T4 PNK

———————–

50µL Total Vol.

Incubate at 37°C for 30 min.

Heat inactivate by heating at 65°C for 20 min.

Notes:

1. 1X T4 DNA Ligase Reaction Buffer contains 1 mM ATP and can be substituted in non-radioactive phosphorylations (T4 PNK exhibits 100% activity in this buffer)
2. Fresh buffer is required for optimal activity (in older buffers, loss of DDT due to oxidation lowers activity)
3. Often, a kinase reaction is followed by a ligation reaction.  In such cases, the T4 PNK reaction is performed in ligase buffer at 37°C for 30 min.  The product of this reaction can be used directly in the ligation reaction without a buffer change or heat inactivation UNLESS there is a need to keep other DNA fragments dephosphorylated during ligation.  When this is desirable, PNK should be heat inactivated prior to ligation.