T3 DNA Ligase

The following reagents are supplied with this product:

NEB #	Component Name	Component #	Stored at (°C)	Amount	Concentration
M0317S     -20       T3 DNA Ligase M0317SVIAL -20 1 x 0.034 ml 3,000,000 units/ml   StickTogether™ DNA Ligase Buffer B0535AVIAL -20 1 x 1 ml 2 X
M0317L     -20       T3 DNA Ligase M0317LVIAL -20 1 x 0.25 ml 3,000,000 units/ml   StickTogether™ DNA Ligase Buffer B0535AVIAL -20 3 x 1 ml 2 X

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

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.

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,

1. Ligation Protocol with T3 DNA Ligase (M0317)

• Activity of DNA Modifying Enzymes in rCutSmart™ Buffer
• Tips for Maximizing Ligation Efficiencies

• DNA Ligase Selection Chart
• Properties of DNA and RNA Ligases
• Substrate-based Ligase Selection Chart

• NEBcloner^®
• NEBioCalculator®

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 Tips for Ligation Reactions

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.

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]

• M0317S_L_v2

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]

• M0317S_L_v1_0021706
• M0317S_L_v1_0021711
• M0317L_v1_10015178
• M0317S_v1_10015180
• M0317L_v1_10026407
• M0317S_v1_10026409
• M0317L_v1_10039217
• M0317S_v1_10039219
• M0317L_v1_10047792
• M0317L_v1_10052788
• M0317L_v1_10059053
• M0317S_v1_10052790
• M0317L_v2_10066721
• M0317S_v2_10083376
• M0317L_v2_10094827
• M0317S_v2_10094830
• M0317L_v2_10108426
• M0317S_v2_10131941
• M0317L_v2_10154519
• M0317S_v2_10164701
• M0317L_v2_10184453
• M0317L_v2_10207425
• M0317S_v2_10205844
• M0317L_v2_10210697
• M0317S_v2_10218710
• M0317L_v2_10231224
• M0317S_v2_10243407
• M0317L_v2_10252945
• M0317L_v2_10256522
• M0317L_v2_10262396
• M0317S_v2_10272307
• M0317L_v2_10272308
• M0317L_v2_10288813
• M0317L_v2_10294627
• M0317L_v2_10306643
• M0317S_v2_10300319
• M0317L_v2_10309625
• M0317L_v2_10311929
• M0317S_v2_10314635
• M0317S_v2_10336824
• M0317L_v2_10319021
• M0317S_v2_10338590

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

• T3 DNA Ligase
• StickTogether™ DNA Ligase Buffer

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.