Description
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:
-
DNA Ligases
- This product can be used in the following applications:
-
Non-Cloning Ligation,
-
DNA Ligation,
-
Cloning Ligation