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

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

Figure 2:  Immobilized T4 DNA Ligase can be re-used 

This product is related to the following categories:

DNA Ligases Products

This product can be used in the following applications:

Non-Cloning Ligation,

DNA Ligation,

Cloning Ligation

Properties & Usage

Reaction Conditions

1X T4 DNA Ligase Reaction Buffer

1 T4 DNA Ligase Reaction Buffer
50 mM Tris-HCl
10 mM MgCl₂
1 mM ATP
10 mM DTT
(pH 7.5 @ 25°C)

Usage Concentration

Storage Buffer

10 mM Tris-HCl
1 mM DTT
0.1 mM EDTA
50 mM KCl
50% Glycerol
pH 7.4 @ -20°C

Heat Inactivation

Unit Assay Conditions

Related Products

Companion Products

• Nuclease-free Water

Product Notes

1. ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA ligase which requires NAD.
2. 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.
3. 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.) 
4. If incubation is longer than 30 minutes, agitation will be required to ensure beads remain in suspension.

Protocols

1. Protocol for Ligation of Fragments with Cohesive Ends using Immobilized T4 DNA Ligase (NEB #M0569)
2. Protocol for Ligation of Fragments with Splinted or Cohesive Ends using Immobilized T4 DNA Ligase (NEB #M0569)

Application Notes

• Generating Oligonucleotide Probes using Immobilized T4 DNA Ligase minimizes sample loss and saves time

Selection Charts

• DNA Ligase Selection Chart
• Properties of DNA and RNA Ligases
• Substrate-based Ligase Selection Chart

Web Tools

• NEBioCalculator®

FAQs

1. How should Immobilized T4 DNA Ligase (NEB #M0569) be stored and handled?
2. How should I mix or resuspend Immobilized T4 DNA Ligase (NEB #M0569) beads prior to use?

Citations & Technical Literature

Quality Assurance Statement

Specifications

• M0569S_L_v1

Certificate Of Analysis

• M0569S_v1_10098368
• M0569S_v1_10143820
• M0569S_v1_10191243
• M0569S_v1_10241822

Safety DataSheets

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Immobilized T4 DNA Ligase

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