Product Class: Other

T4 Polynucleotide Kinase
NEBU cloned at NEB recombinant 37 65 Heat

Product Introduction

  • 5' phosphorylation of DNA/RNA for subsequent ligation
  • End labeling DNA or RNA for probes and DNA sequencing
  • Removal of 3' phosphoryl groups
 
Catalog # Size Concentration
M0201S 500.0 units 10000 units/ml
M0201L 2500.0 units 10000 units/ml

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Product Information

Description

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. Polynucleotide Kinase also catalyzes the removal of 3´-phosphoryl groups from 3´-phosphoryl polynucleotides, deoxynucleoside 3´-monophosphates and deoxynucleoside 3´-diphosphates (1).

Product Source

A E. coli strain that carries the cloned T4 Polynucleotide Kinase gene. It is purified by a modification of the method of Richardson (1).
This product is related to the following categories:
DNA Labeling Products,
Kinases Products,
This product can be used in the following applications:
Phosphorylation (Kinase)

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Unit Definition

One unit of enzyme catalyzes the phosphorylation of 20 pmol of fluorescently labeled oligo in 30 min at 37˚C.

Reaction Conditions

1X T4 Polynucleotide Kinase Reaction Buffer
Incubate at 37°C

1X T4 Polynucleotide Kinase Reaction Buffer
70 mM Tris-HCl
10 mM MgCl2
5 mM DTT
(pH 7.6 @ 25°C)

Usage Concentration

10,000 units/ml

Storage Buffer

10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
0.1 µM ATP
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 minutes

Application Features

  • End-labeling DNA or RNA for probes and DNA sequencing (2)
  • Addition of 5´-phosphates to oligonucleotides to allow subsequent ligation
  • Removal of 3´-phosphoryl groups (3)

Product Notes

  1. Gaps can be phosphorylated with elevated levels of ATP. Nicks are not phosphorylated efficiently. CTP, GTP, TTP, UTP, dATP or dTTP can be substituted for ATP as a phosphate donor (1).
  2. Dephosphorylation prior to end-labeling can be avoided by utilizing the exchange reaction, although this results in lower specific activity labeling.
  3. Sufficient incorporation levels can be attained using the supplied buffer supplemented with 100 µM ADP in the final reaction. However, higher levels of incorporation with the exchange reaction are achieved when using the buffer described in (2).

References

  1. Richardson, CC. (1981). The Enzyme. 14, 299-314.
  2. Sambrook, J. et al. (1989). Molecular Cloning: A Laboratory Manual.. 10.59-10.67,11.31-11.33.
  3. Cameron, V. and Uhlenbeck, O.C. (1977). Biochemistry. 16, 5120-5126.
  4. Berkner, K.L. and Folk, W.R. (1977). J. Biol. Chem.,. 252, 3176-3184.
  5. Soltis, D.A. and Uhlenbeck, 0.C. (1982). J. Biol. Chem.. 257, 11332-11339.
  6. Wang, L.K. and Human, S. (2001). J. Biol. Chem.. 276, 26868-26874.
  7. Wang, L.K. and Shuman, S. (2002). Nucl. Acids Res.. 30, 1073-1080.
  8. Vance, J.R. and Wilson, T.E. (2001). Mol. Cell. Biol. 21, 7191-7198.
  9. Interthal, H. et al. (2005). J. Biol. Chem.,. 280, 36518-36528.

Protocols, Manuals & Usage

Protocols

  1. Radioactive Labeling with T4 PNK or T4 PNK (3´ phosphatase minus)
  2. Non-radioactive Phosphorylation with T4 PNK or T4 PNK (3´ phosphatase minus)
  3. End-labeling Protocol

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. What factors can cause incomplete phosphorylation when using T4 Polynucleotide Kinase?
  2. Can I use T4 Polynucleotide Kinase and T4 DNA Ligase in the same reaction buffer?
  3. How can the rate of phosphorylation be improved when using T4 Polynucleotide Kinase?
  4. Do I need to dephosphorylate prior to labeling?
  5. How much substrate can be phosphorylated in a standard reaction?
  6. How many units of T4 Polynucleotide Kinase should be used for a typical reaction?
  7. How do I inactivate the enzyme?
  8. How to calculate the molarity of ends?
  9. What labels can be used?
  10. Will T4 Polynucleotide Kinase work in rCutSmart Buffer?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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