T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5´ phosphate and 3´ hydroxyl termini in duplex DNA or RNA. T4 DNA Ligase joins blunt end and cohesive end termini as well as repairs single stranded nicks in duplex DNA and some DNA/RNA hybrids (1).
Immobilized T4 DNA Ligase (IM T4 DNA Ligase) is a slurry of magnetic beads coated with T4 DNA Ligase to produce a 10 mg/ml solution (50% glycerol) with an effective concentration of 60 cohesive end units (CEU) per microliter of slurry. Following a reaction, enzyme can be removed using a magnet, and can be re-used.
Relative Activity of Immobilized T4 DNA Ligase at different temperatures
Activity of Immobilized T4 DNA Ligase in Different NEB Buffers
NEBuffer r 2.1
Phi29 DNA Polymerase Buffer
Standard Taq Buffer
Q5 Reaction Buffer
T4 DNA Ligase Buffer
NEBNext Quick Ligation Buffer
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ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA ligase which requires NAD.
If IM T4 DNA Ligase is less than 10% of the total reaction volume, set-up reaction in the following order: H2O, buffer, DNA and finally enzyme.
If IM T4 DNA Ligase is more than 10% of the total reaction volume, we recommend setting up the reaction as follows: Prepare the substrate in 1X reaction buffer. Add IM T4 DNA Ligase into a fresh tube. Put the tube on a magnet for 3 minutes to pellet the beads. Then carefully remove the supernatant from the bead pellet and add the substrate mixture to the bead pellet. (Do not remove the supernatant until you are ready to assemble the reaction, as the beads should not be allowed to dry out.)
If incubation is longer than 30 minutes, agitation will be required to ensure beads remain in suspension.
Engler, M.J. and Richardson, C.C. (1982). P.D. Boyer(Ed.), 5, 3. San Diego: Academic Press.
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Specifications & Change Notifications
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