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.
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