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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
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