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T4 DNA Ligase
Gray property icon for NEBU recombinant 16 Gray property icon for heat inactivation 65°

Looking for additional T4 DNA Ligase formulations and variants?  Learn More

Product Introduction

T4 DNA Ligase will ligate these substrates:

dsDNA

dsDNA substrate for ligase selection:5´ [ straight line ] OH p [ straight line ] 3´3´ [ straight line ] p OH [ straight line ] 5´

dsDNA substrate for ligase selection:5´ [ straight line ] A-OH p [ straight line ] 3´3´ [ straight line ] p OH-T [ straight line ] 5´

dsDNA substrate for ligase selection:5´ [ straight line ] OH p [ straight line ] 3´3´ [ straight line ] p OH [ straight line ] 5´

dsDNA substrate for ligase selection:5´ [ straight line ] OH p [ straight line ] 3´3´ [ straight line ] p OH [ straight line ] 5´ 

Nicked DNA/RNA

Nicked substrate for ligase selection:5´ [ straight line ] OH p [ straight line ] 3´3´ [ straight line ] 5´

Nicked substrate for ligase selection:5´ [ wavy line ] OH p [ straight line ] 3´3´ [ straight line ] 5´


Ligase substrate key with [ black straight line ] DNA and [ red wavy line ] RNA 

 

  • Catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA.
  • This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA and some DNA/RNA hybrids (1).
  • The industry standard for performance and quality
  • Applicable for use in high-complexity library cloning
  • Not sure which ligase to choose? Refer to our DNA and RNA Ligase Properties Chart or DNA Ligase Selection Chart
  • Available GMP grade. Learn more.
  • Available glycerol-free. Learn more.
Bioz Badge Exists : True
Catalog # Size Concentration
M0202S 20000 units 400000 units/ml
M0202T 20000 units 2000000 units/ml
M0202L 100000 units 400000 units/ml
M0202M 100000 units 2000000 units/ml

Featured Videos

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    Quick Tips - Do I need a 5' phosphate for ligation?

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    NEBioCalculator® - Using the Ligation module

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    Quick Tips - Electroporation after ligation

  • NEBTV_DNA-Modifying-Enzymes-and-Cloning-Techniques_1920

    Quick Tips - How do I calculate how much DNA to add to a ligation reaction?

  • NEBTV_DNA-Modifying-Enzymes-and-Cloning-Techniques_1920

    Quick Tips - Troubleshooting problematic ligation reactions

  • NEBTV_DNA-Modifying-Enzymes-and-Cloning-Techniques_1920

    Quick Tips - Why is there precipitate in my T4 DNA ligase buffer?

  • NEBTV_DNA-Modifying-Enzymes-and-Cloning-Techniques_1920

    Quick Tip - What is the ideal incubation temperature for ligation?

  • NEBTV_DNA-Modifying-Enzymes-and-Cloning-Techniques_1920

    Quick Tip - When is a long incubation time necessary for ligation?

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Product Information

Description

Highlights

  • Isolated from a recombinant source
  • Supplied with 10X Reaction Buffer
For details on NEB's quality controls for DNA ligases, visit our Ligase Quality page.

Figure 1: T4 DNA Ligase competitor study – nuclease contamination 
Bar graph of nuclease contamination in NEB lots of T4 DNA Ligase versus lots from other companies
T4 DNA Ligase from multiple suppliers was tested in reactions containing a fluorescent labeled single stranded, double stranded blunt, 3’overhang or 5’ overhang containing oligonucleotides. The percent degradation by contaminating nucleases is determined by capillary electrophoresis and peak analysis. The resolution is at the single nucleotide level.

Product Source

An E. coli strain that carries the cloned T4 DNA Ligase gene.
This product is related to the following categories:
DNA Ligases
This product can be used in the following applications:
BioBrick® Assembly,
Cloning Ligation,
NEBridge® Golden Gate Assembly

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

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

Properties & Usage

Materials Required but not Supplied

Unit Definition

One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer.

Concentration:
400,000 cohesive end units/ml and 2,000,000 cohesive end units/ml

Reaction Conditions

1X T4 DNA Ligase Reaction Buffer
Incubate at 16°C

1X T4 DNA Ligase Reaction Buffer
50 mM Tris-HCl
10 mM MgCl2
1 mM ATP
10 mM DTT
(pH 7.5 @ 25°C)

Storage Buffer

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

Heat Inactivation

65°C for 10 minutes

Application Features

  • Cloning of restriction fragments
  • Joining linkers and adapters to blunt-ended DNA

Product Notes

  1. ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA ligase which requires NAD.
  2. To dilute T4 DNA Ligase that will subsequently be stored at -20°C, 50% glycerol storage buffer (Diluent Buffer A (with rAlbumin), NEB #B8532S) should be used; to dilute for immediate use, 1X T4 DNA Ligase Reaction Buffer can be used.
  3. Ligation can also be performed in any of the four restriction endonuclease NEBuffers or in T4 Polynucleotide Kinase Buffer if they are supplemented with 1 mM ATP.
  4. Room Temperature Ligation
    For convenience, ligations may be done at room temperature (20-25°C). For cohesive (sticky) ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 10 minutes. For blunt ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 2 hours or 1 µl high concentration T4 DNA Ligase for 10 minutes. Alternatively, NEB's Quick Ligation Kit (#M2200S) [30 reactions] or (#M2200L) [150 reactions]) is uniquely formulated to ligate both blunt and cohesive (sticky) ends in 5 minutes at room temperature.

References

  1. Engler, M.J. and Richardson, C.C. (1982). P.D. Boyer(Ed.), 5, 3. San Diego: Academic Press.
  2. Remaut, E., Tsao, H. and Fiers, W. (1983). Gene. 22, 103-113.
  3. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd Ed.). 1.53-1.73.

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. What are some potential problems with the ligation reaction using T4 DNA Ligase that can lead to transformation failure?
  2. What problems can be encountered in the restriction digest that can cause ligation using T4 DNA Ligase or subsequent transformation to fail?
  3. What controls should be run to test the cells and DNA when using T4 DNA Ligase?
  4. When should T4 DNA Ligase be the enzyme of choice?
  5. Can the T4 DNA Ligase be used with the Quick Ligase buffer?
  6. What is the definition of a Weiss Unit and a Cohesive End Unit?
  7. What is the difference between the two definitions and why does NEB use the Cohesive End Unit?
  8. How much DNA should be used in a ligation using T4 DNA Ligase?
  9. Can T4 DNA Ligase be used in other NEBuffers, including rCutSmart?
  10. Can T4 DNA Ligase be heat inactivated?

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.

Specification Change Notifications

To request a specification sheet for a custom product not available below please send a request to custom@neb.com.

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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