T4 POLYNUCLEOTIDE KINASE
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T4 Polynucleotide Kinase
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.
Key Features and Benefits
- 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 | ≥ 99% |
| dsDNA Exonuclease* | <1% released |
| ssDNA Exonuclease* | <1% released |
| DNA contamination Assay ( E. coli , mammalian, library)* | < 10 copies |
| Phosphatase Contamination* | < 1% released |
| Endonuclease Contamination* | 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
*Please reference our technical guide for instructions for use.
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
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Custom Genomic Solutions Brochure
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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 |
| 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 | |
|---|---|
| Protein Purity | ≥ 99% |
| dsDNA exonuclease* | <1% released |
| ssDNA exonuclease* | <1% released |
| DNA contamination* (E. coli, mammalian, library) | <10 copies |
| Phosphatase Contamination* | <1% released |
| Endonuclease Contamination* | 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 relevant 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
- Preparation of 5' and 3' DNA substrate ends for homebrew library preparation
What is the storage buffer for T4 PNK?
Please see the Technical Guide for more information on T4 Polynucleotide Kinase storage buffer.
Enzyme Storage Buffer: 10 mM Tris-HCl pH 7.4, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50% Glycerol, 0.1 μM ATP
Is the reaction buffer supplied with T4 PNK?
The T4 Polynucleotide Kinase can be supplied with or without the reaction buffer. The T4 Polynucleotide Kinase Kit (10 U/μL) contains a 10X T4 PNK Reaction Buffer. Please see the Technical Guide for more information on the recommended reaction buffer composition.
10X T4 PNK Reaction Buffer: 700 mM Tris-HCl, pH 7.6 at 25°C, 100 mM MgCl2 , 50 mM DTT
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, de-phosphorylation prior to labeling is not required. However, dephosphorylating can increase label incorporation.
What is the optimal temperature for T4 PNK?
T4 Polynucleotide Kinase exhibits maximal phosphorylation activity at 37°C.
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.
Where can I go for additional help or technical support?
If you need more detailed guidance or have questions not addressed here, please reach out to our support team at support@watchmakergenomics.com or submit a request via our Technical Support form. Please include details such as product name, lot number, version of protocol you’re using, and a brief description of your question or issue so we can assist you promptly.
Do you offer custom formats?
Yes! Watchmaker offers custom fill volumes, 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 processes enable rapid turnaround time on customization. Please contact sales@watchmakergenomics.com or submit a request via our Custom Solutions form 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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