T4 POLYNUCLEOTIDE KINASE
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T4 PNK Highlights
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.
- Prepare DNA for ligation through 5' DNA phosphorylation and 3' DNA dephosphorylation
- Reduce material costs with a drop-in solution that delivers equivalent performance at a differentiated price-point
- Custom formats available, including high concentration
- Highly stringent enzyme manufacturing ensures quality performance across lots
Applications
- NGS library preparation
- Cloning (5'-phosphorylation of DNA and RNA prior to ligation)
- Removal of 3'-phosphates
- 5'-labeling of DNA and RNA to be used as probes, primers, and markers
QC Specifications
| Description | Specification |
|---|---|
| Protein Purity Assay | ≥ 99% |
| dsDNA Exonuclease Assay* | <1% released |
| ssDNA Exonuclease Assay* | <1% released |
| DNA contamination Assay ( E. coli , mammalian, library)* | < 10 copies |
| Phosphatase Contamination Assay* | < 1% released |
| Endonuclease Contamination Assay* | Not detectable |
*As assessed using 250 U of enzyme input per assay.
Properties
Unit definition: One unit of T4 Polynucleotide Kinase 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
Reaction conditions:
1X T4 PNK Reaction Buffer
Incubate at 37°C
Storage buffer: 10mM Tris-HCl, pH7.4, 50mM KCl, 0.1 EDTA, 1mM DTT, 50% Glycerol, 0.1 µM ATP
1X T4 PNK Reaction Buffer: 70 mM Tris-HCl, pH 7.6 at 25°C, 10 mM MgCl₂, 5 mM DTT
Heat inactivation: 65°C for 20 min
Molecular weight: 34.6 kDa
BROCHURES
Precision Enzymes and Proteins Brochure
Custom Genomic Solutions Enzymes MDx DNA Polymerase I Equinox Library Amplification Equinox Uracil-Tolerant Library Amplification phi29 DNAP RNase Inhibitor StellarScript RT StellarScript HT RT StellarScript HT+ RT StellarTaq DNA Polymerase T4 DNA Ligase T4 DNAP T4 Polynucleotide Kinase T4 Gene 32 Protein Taq DNAPWatchmaker Genomics offers a portfolio of precision-engineered enzymes and proteins, designed to meet the demands of high-stringency applications. We provide tailored services such as custom fills, formats, packaging, and labeling to accommodate unique specifications.
PROTOCOLS
T4 Polynucleotide Kinase Technical Guide
Enzymes T4 Polynucleotide KinaseProtocol for T4 Polynucleotide Kinase (10 U/µL) which transfers the γ-phosphate from ATP to the 5'-OH of DNA or RNA, enabling 5'-end labeling and phosphorylation for ligation. It also removes 3'-phosphates.
| CONFIGURATION | |
|---|---|
| Kit contents | T4 Polynucleotide Kinase (10 U/µL) with or without 10X T4 PNK Reaction Buffer |
| Unit definition | One unit of T4 Polynucleotide Kinase 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 |
| Enzyme storage buffer | 10mM Tris-HCl, pH7.4, 50mM KCl, 0.1 EDTA, 1mM DTT, 50% Glycerol, 0.1 µM ATP |
| 10X T4 PNK Reaction Buffer: | 700 mM Tris-HCl, pH 7.6 at 25°C, 100 mM MgCl₂, 50 mM DTT |
| QUALITY CONTROL | |
|---|---|
| Purity (SDS-PAGE) | ≥ 99% |
| dsDNA exonuclease* | <1% released |
| ssDNA exonuclease* | <1% released |
| DNA contamination Assay* (E. coli, mammalian, library) | <10 copies |
| Phosphatase Contamination Assay* | <1% released |
| Endonuclease Contamination Assay* | Not detectable |
*As assessed using 250 U of enzyme input per assay.
| SHIPPING AND HANDLING | |
|---|---|
| Shipping conditions | Ice packs |
| Storage | -20°C ± 5°C |
| Shelf life (off-the-shelf products) | ≥ 12 months |
| Shelf life (custom products) | ≥ 18 months |
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?
T4 PNK is supplied with a 10X reaction buffer as follows:
- 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.
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.
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| Description | 2.5 kU | |
|---|---|---|
| T4 Polynucleotide Kinase Kit (10 U/µL) incl. 10X T4 PNK Reaction Buffer | 7K0046-250UL | Request a quote |
| T4 Polynucleotide Kinase (10 U/µL) | 7K0018-250UL | Request a quote |
Please contact sales@watchmakergenomics.com to inquire about custom kit configurations.
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