For details on NEB's quality controls for DNA ligases, visit our Ligase Quality page.
A thermotolerant variant of T4 DNA Ligase, Hi-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 temperatures than wild type T4 DNA Ligase. Hi-T4 DNA Ligase will join blunt end and cohesive end termini as well as repair single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids at temperatures as high as 50ºC.
Figure 1: Hi-T4 DNA Ligase displays increased thermotolerance compared to T4 DNA Ligase.
400 units of T4 DNA Ligase (NEB #M0202) or Hi-T4 DNA Ligase (NEB #M2622) were pre-incubated at 45°C in 1X T4 DNA Ligase Buffer in the absence of DNA substrate for the indicated time. Residual ligase activity was tested by adding 0.12 μM HindIII (cohesive end) fluorescently-labeled fragments and incubating at 37°C for 15 minutes. Ligation products were detected by capillary electrophoresis.
T4 DNA Ligase steadily loses activity at 45°C, whereas Hi-T4 DNA Ligase retains full activity on a cohesive end substrate, even after 72 hours at 45°C.
Figure 2: Hi-T4 DNA Ligase withstands higher temperature cycling than T4 DNA Ligase.
1,000 units of T4 DNA Ligase (NEB #M0202) or Hi-T4 DNA Ligase (NEB #M2622) in (1X) T4 DNA Ligase Buffer were cycled in the presence of DNA at 42°C (A) or 50°C (B) for 5 minutes followed by 16°C for 5 minutes in the presence of 1 μg pBR322. Following the indicated number of cycles (0, 15, 30, 45 or 60), ligase activity was tested on a fluorescently-labeled, cohesive-end substrate (HindIII overhang, 25°C for 10 minutes). Ligation products were detected by capillary electrophoresis. Remaining ligase activity (relative to 0 cycle data point) is shown.
A. T4 DNA Ligase gradually loses activity after cycling between 16°C and 42°C (gold line, A), while Hi-T4 DNA Ligase maintains activity after 60 cycles between 16°C and 42°C (orange line, A).
B. T4 DNA Ligase is completely inactivated after only 15 cycles between 16°C and 50°C (gold line, B), while Hi-T4 DNA Ligase maintains ~60% activity after 60 cycles between 16°C and 50°C (orange line, B).
Highlights
Engineered thermotolerant variant of T4 DNA Ligase
Isolated from a recombinant source
Product Source
An E. coli strain that carries a plasmid encoding the engineered Hi-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 µl.
Reaction Conditions
1X T4 DNA Ligase Reaction Buffer
Incubate at 25°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
ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA ligase which requires NAD.
To dilute Hi-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 cohesive (sticky) ends (1X T4 DNA Ligase Buffer), incubate 1 µl of Hi-T4 DNA Ligase in a 20 µl reaction between 25-50ºC for 10 minutes. For blunt ends or single base overhangs (1X StickTogether DNA Ligase Buffer), incubate 1 µl of Hi-T4 DNA Ligase in a 20 µl reaction between 25-50ºC 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.
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Specifications & Change Notifications
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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