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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
Temperature
% Activity
16ºC
100%
25ºC
100%
37ºC
50%
Activity of Immobilized T4 DNA Ligase in Different NEB Buffers
Buffer
pH
% Activity
NEBuffer r1.1
7.0
50%
NEBuffer r 2.1
7.9
100%
NEBuffer r3.1
7.9
70%
rCutSmart Buffer
7.9
100%
Phi29 DNA Polymerase Buffer
7.5
100%
Standard Taq Buffer
8.3
70%
Thermopol Buffer
8.8
70%
Q5 Reaction Buffer
8.5
10%
T4 DNA Ligase Buffer
7.5
100%
NEBNext Quick Ligation Buffer
7.6
25%
Figure 1: Successful ligation of Lambda DNA- HindIII Digest by Immobilized T4 DNA Ligase
Ligations of Lambda DNA- HindIII Digest (NEB #N3012) were performed with either no ligase, soluble T4 DNA Ligase (NEB #M0202) or Immobilized T4 DNA Ligase (NEB #M0569) according to recommended protocols. Results indicate ligation is equivalent between soluble and immobilized enzyme.
Figure 2: Immobilized T4 DNA Ligase can be re-used
Consecutive splint ligations of Nanopore Adaptors were performed with Immobilized T4 DNA Ligase (NEB #M0569) in Quick Ligation Buffer. The data shows that activity remains unaffected after 20 consecutive ligation reactions.
This product is related to the following categories:
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.
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]
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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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.
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