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Cat. No.: EDL-200 (for 200U)

Cat. No.: EDL-1k (for 1KU)

Cat. No.: EDL-5k (for 5KU)

Description

E. coli DNA Ligase is a coenzyme-dependent enzyme that utilizes NAD+ (nicotinamide adenine dinucleotide) and requires Mg2+ as a cofactor. It catalyzes the formation of a phosphodiester bond between the 5' phosphate and 3' hydroxyl groups in double-stranded DNA, thereby mediating the ligation of cohesive ends or repairing nicks in double-stranded DNA.

E. coli DNA Ligase can catalyze the formation of phosphodiester bonds between adjacent 5' phosphate and 3' hydroxyl groups in cohesive ends of double-stranded DNA. However, under standard conditions, it cannot ligate blunt-ended double-stranded DNA. By adding 10-15% PEG (polyethylene glycol) and an appropriate high concentration of monovalent cations, it is also possible to catalyze the ligation of blunt-ended double-stranded DNA. E. coli DNA Ligase exhibits activity within a certain temperature range (4-37°C) and can be inactivated by heat (incubation at 65°C for 20 minutes).

Applications

Ligation of cohesive-ended double-stranded DNA molecules; repair of nicks in double-stranded DNA; cloning of cDNA after second-strand synthesis.

Source

Purified from recombinant E. coli strains carrying the gene encoding E. coli DNA Ligase.

Activity Definition

One unit is defined as the amount of enzyme required to achieve 50% ligation of HindIII fragments of λ DNA (5' DNA termini concentration of 0.12μM, 300μg/ml) in a total reaction volume of 20μl in 30 minutes at 16°C using 1X E. coli DNA Ligase Reaction Buffer.

Purity

Free from other types of DNA ligases, endonucleases, exonucleases, ribonucleases, and phosphatases other than E. coli DNA Ligase.

Enzyme Storage Buffer

10mM Tris-HCl, 50mM KCl, 1mM DTT, 0.1mM EDTA, 200µg/ml BSA, 50% Glycerol (pH 7.4 @ 25°C).

10X Reaction Buffer

300mM Tris-HCl, 40mM MgCl2, 260μM NAD, 10mM DTT, 500μg/ml BSA (pH 8.0 @ 25°C).

Inactivation or Inhibition

E. coli DNA Ligase can be inactivated by incubation at 65°C for 20 minutes.

Precautions

• E. coli DNA Ligase requires NAD (nicotinamide adenine dinucleotide) as a coenzyme, unlike T4 DNA ligase, which uses ATP as a coenzyme.
• The efficiency of E. coli DNA Ligase for blunt-ended fragment ligation is extremely low. For blunt-end ligation, it is recommended to use T4 DNA Ligase.
• E. coli DNA Ligase catalyzes the ligation of double-stranded DNA molecules and cannot be used for the ligation of single-stranded DNA or RNA.
• The ligation reaction of E. coli DNA Ligase requires NAD as a cofactor. The 10X Reaction Buffer contains NAD as a cofactor, so it is recommended to store the 10X Reaction Buffer at -80°C for long-term storage to extend the half-life of NAD.
• This product is intended for scientific research purposes by professionals only. It should not be used for clinical diagnosis or treatment, food or drug purposes, and should not be stored in regular residential areas.
• For your safety and health, please wear appropriate laboratory attire and disposable gloves when handling this product.

Storage

The minimum shelf life is 2 years at -20°C. 10X Reaction Buffer is recommended to be stored at -80°C for long-term storage to extend the half-life of NAD.

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• T7 DNA Ligase
• E. coli DNA Ligase
• PBCV-1 DNA Ligase
• T3 DNA Ligase
• Taq DNA Ligase
• Pfu DNA Ligase
• High-Fidelity DNA Ligase
• High-Salt DNA Ligase

SBS Genetech is recognized as one of the global major leading industry players in Ligase Market by third-party market researchers. For more details, please visit Ligase Market - A Global and Regional Analysis Focus on Product, Source, Application, End User, and Country - Analysis and Forecast, 2022-2032