For details on NEB's quality controls for DNA ligases, visit our Ligase Quality page.
A salt tolerant variant of T4 DNA Ligase, Salt-T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5´ phosphate and 3´ hydroxyl termini in duplex DNA or RNA and is designed to function at higher salt concentrations than wild type T4 DNA Ligase. This enzyme will join cohesive end termini at salt concentrations as high as 300 mM without any loss in activity. This enzyme is insensitive to salt carried over from other reaction components (vector or insert DNA) and allows ligation reactions to proceed in alternative reaction buffers with higher levels of salt (e.g., NEBuffer 3.1).
Figure 1: Salt-T4 DNA Ligase displays increased salt tolerance on a cohesive end substrate as compared to T4 DNA Ligase.
400 units of T4 DNA Ligase (NEB #M0202) or Salt-T4 DNA Ligase (NEB #M0467) was incubated with 0.12 μM HindIII (cohesive end) fluorescent-labeled fragments at 25°C for 10 minutes in the presence of 1X T4 DNA Ligase Buffer containing increasing amounts of salt (0-1000 mM NaCl). Ligation products were detected by capillary electrophoresis.
T4 DNA Ligase rapidly loses activity at salt concentrations above 200 mM, whereas Salt-T4 DNA Ligase retains > 60% activity on a cohesive end substrate at salt concentrations as high as 500 mM.
Product Source
An E. coli strain that carries a plasmid encoding the engineered Salt-T4 DNA Ligase gene.
This product is related to the following categories:
One unit is defined as the amount of enzyme required to give 50% ligation of 6 µg of Lambda-HindIII DNA in 30 minutes at 25°C in a total reaction volume of 20 µ in 1X T4 DNA Ligase Reaction Buffer supplemented with 100 mM NaCl.
Reaction Conditions
1X T4 DNA Ligase Reaction Buffer
Incubate at 25°C
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
ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA ligase which requires NAD.
To dilute Salt-T4 DNA Ligase that will subsequently be stored at -20°C, 50% glycerol storage buffer (Diluent Buffer A, NEB #B8001S) should be used; to dilute for immediate use, 1X T4 DNA Ligase Reaction Buffer can be used.
Ligation Temperature
For convenience, ligations may be done at room temperature (20-25°C). For cohesive (sticky) ends (1X T4 DNA Ligase Buffer), use 1 µl of Salt-T4 DNA Ligase in a 20 µl reaction for 10 minutes. For blunt ends or single base overhangs (1X StickTogether™ DNA Ligase Buffer), use 1 µl of Salt-T4 DNA Ligase in a 20 µl reaction for 10 minutes. Do not heat kill the reaction because heat treating the PEG in the StickTogether™ DNA Ligase Buffer will inhibit transformation.
References
Engler, M.J. and Richardson, C.C. (1982). P.D. Boyer(Ed.), 5, San Diego: Academic Press.
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 & Change Notifications
Effective June 16, 2022, buffer volume will change to 1.0 ml.
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]
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]
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