Product Class: Other

T3 DNA Ligase
NEBU cloned at NEB recombinant 25 No

Product Introduction

T3 DNA Ligase will ligate these substrates:

dsDNA

(1)

Nicked DNA/RNA






1. Note: Trace amounts of this substrate can be ligated with overnight incubation, and ligation is further enhanced in Stick Together DNA Ligase Buffer.  Please note that Blunt TA Ligase Mastermix is the recommended ligase product for this substrate.

 

Catalog # Size Concentration
M0317S 100000.0 units 3000000 units/ml
M0317L 750000.0 units 3000000 units/ml

Product Information

Description

T3 DNA Ligase is an ATP-dependent ds DNA ligase from bacteriophage T3. It will catalyze the formation of a phosphodiester bond between adjacent 5´ phosphate and 3´ hydroxyl groups of duplex DNA. Cohesive ends, blunt ends, and nick sealing can all be efficiently catalyzed by T3 DNA Ligase (1). As with T4 DNA Ligase, blunt end ligation is enhanced by the addition of PEG 6000 to the reaction (2). T3 DNA Ligase exhibits a higher tolerance (2-fold) for NaCl in the reaction compared to T4 DNA Ligase, making the enzyme a versatile choice for in vitro molecular biology protocols requiring DNA ligase activity.

Product Source

An E. coli strain containing a recombinant gene encoding T3 DNA Ligase.
This product is related to the following categories:
DNA Ligases Products
This product can be used in the following applications:
Cloning Ligation

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 is defined as the amount of enzyme required to give 50% ligation of 100 ng HindIII fragments of λ DNA in a total reaction volume of 20 μl in 1 minute at 25°C in 1X StickTogether DNA Ligase Buffer.

Reaction Conditions

1X StickTogether™ DNA Ligase Buffer
Incubate at 25°C

1X StickTogether™ DNA Ligase Buffer
66 mM Tris-HCl
10 mM MgCl2
1 mM ATP
1 mM DTT
7.5% Polyethylene glycol (PEG 6000)
(pH 7.6 @ 25°C)

Storage Buffer

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

Heat Inactivation

No

Application Features

  • Cloning of DNA fragments generated by restriction enzyme digestion
  • Cloning of PCR products
  • Adding linkers or adapters to dsDNA
  • Circularization of linear DNA
  • Nick-sealing in dsDNA
  • Site-directed mutagenesis

Product Notes

  1. ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA Ligase which requires NAD.
  2. T3 DNA Ligase is also active in buffers without PEG 6000, such as our T4 DNA Ligase Buffer and NEBuffers, for applications in which PEG 6000 is detrimental. Please remember to supplement the reaction with 1 mM ATP (final concentration). In these buffers T3 DNA Ligase exhibits an approximately 10-fold reduction in activity. In applications where a high concentration of NaCl needs to be maintained, we suggest using a reaction buffer without PEG 6000.
  3. Standard vector + insert ligations in 10–20 μl reaction volumes are usually performed for 30 minutes at 25°C.
  4. Dilution of enzyme for long-term storage at -20°C should be performed with the storage buffer containing 50% glycerol. Diluent A (NEB #B8001) can also be used for those application in which BSA, present in Diluent A, will not interfere.
  5. Heating a reaction containing T3 DNA Ligase at 65°C for 10 minutes will inactivate the enzyme. However, the reaction needs to be performed in a buffer without PEG. Do not heat inactivate if there is PEG in the reaction buffer, as transformation will be inhibited.

References

  1. Cai, L. et al. (2004). J. Biochem. 135, 397-403.
  2. Bauer RJ, Zhelkovsky A, Bilotti K, Crowell LE, Evans TC Jr, McReynolds LA, et al. (2017). Comparative analysis of the end-joining activity of several DNA ligases. PLoS ONE. 12(12): e0190062., DOI: 0190062,

Protocols, Manuals & Usage

Protocols

  1. Ligation Protocol with T3 DNA Ligase (M0317)

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. What type of DNA ends can T3 DNA Ligase ligate?
  2. Can T3 DNA Ligase be used with a buffer that does not contain PEG?
  3. Does T3 DNA Ligase have a higher tolerance to salt than T4 DNA Ligase?
  4. Can T3 DNA Ligase be heat inactivated?
  5. What are some potential problems with the ligation reaction using T3 DNA Ligase that can lead to transformation failure?
  6. What are some other problems that should be considered when trouble shooting a transformation problem?
  7. What problems can be encountered in the restriction digest that can cause ligation using T3 DNA Ligase or subsequent transformation to fail?
  8. What controls should be run to test the cells and DNA when using T3 DNA Ligase?
  9. How much DNA should be used in a ligation using T3 DNA Ligase?
  10. Will T3 DNA Ligase work in rCutSmart Buffer?

Troubleshooting

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 [email protected].

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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