What is T4 Polynucleotide Kinase (PNK)?
T4 Polynucleotide Kinase (T4 PNK) catalyzes the transfer of the terminal gamma-phosphate from ATP to the 5′-OH group of double- and single-stranded DNA, RNA and nucleoside 3′-monophosphate molecules. T4 PNK also exhibits 3′-phosphatase activity and 5′-ADP phosphatase activity.
What is the unit definition of T4 PNK?
One unit of T4 PNK is defined as the amount of enzyme catalyzing the incorporation of 1 nmol of phosphate onto a DNA substrate from an ATP donor in 30 minutes at 37°C.
What are the applications for T4 PNK?
- Radioactive or non-radioactive labeling of 5′-termini of nucleic acids (i.e., probes, primers, or markers)
- 5′-phosphorylation of nucleic acid substrates for downstream use in ligation
- Removal of 3′-phosphate groups
What is the storage buffer for T4 PNK?
- 10 mM Tris-HCl pH 7.4
- 50 mM KCl
- 0.1 mM EDTA
- 1 mM DTT
- 50% Glycerol
- 0.1 μM ATP
What is the recommended reaction buffer for T4 PNK?
- 700 mM Tris-HCl
- 100 mM MgCl₂
- 50 mM DTT pH 7.6 @ 25°C
What steps can be taken to ensure complete phosphorylation?
- Add fresh DTT to the reaction buffer. DTT can oxidize after time and an active reducing agent is required for optimal T4 PNK activity
- Remove excess salt prior to phosphorylation
- If the ends are blunt or the 5′ is recessed, heat the substrate for 10 minutes at 70°C and incubate on ice before adding the enzyme and reaction buffer
- Remove or heat inactivate any phosphatase
Do I need to dephosphorylate prior to labeling?
No, but it does increase incorporation to dephosphorylate first.
How is T4 PNK inactivated?
Incubation at 65°C for 20 minutes will inactivate T4 PNK.
What conditions can be used for labeling of the 5' termini?
Please reference our technical guide for instructions for use.
1. On ice, combine components as specified:
| Component | Final quantity (for 50 µL reaction) |
|---|---|
| DNA | Up to 300 pmol 5′ termini |
| 10X T4 PNK reaction buffer | 5 µL |
| ATP (10 mM) | 50 pmol of ATP |
| T4 PNK (10 U/µL) | 2 µL (20 U) |
| Nuclease free water | up to 50 µL |
2. Incubate at 37°C for 30 minutes.
3. Heat inactivate by incubating at 65°C for 20 minutes.
Note: For radio labeling [³³P] ATP may be substituted for ATP and the maximum amount of DNA should be no more than 50 pmol.
What is the extinction coefficient for T4 PNK?
The extinction coefficient for T4 PNK is 1.873 (mL/(mg*cm). If measuring protein concentration (A280), we recommend you use the protein’s extinction coefficient to ensure an accurate reading.
Do you offer custom formats?
Yes! Watchmaker offers customer fills, packaging, concentrations, and labeling – including private label – designed to meet your unique needs. We offer flexible terms to serve organizations of any size, and our right-sized processes enable rapid turnaround time on customization. Please contact sales@watchmakergenomics.com to learn more about our capabilities.
Are you ISO 13485-certified?
Yes! Our Quality Management System has achieved ISO 13485:2016 Certification. Our certificate has been awarded for the design, development, manufacture, contract manufacture, and support of high performing reagents for genomics applications in medical research. Download the certificate here.