E. coli DNA Ligase produced by Aladdin is a recombinant enzyme expressed and purified using our independently developed technical platform.E. coli DNA Ligase is an NAD⁺ (nicotinamide adenine dinucleotide)-dependent and Mg²⁺-dependent DNA ligase that catalyzes the formation of a phosphodiester bond between the 5'-phosphate and 3'-hydroxyl groups in double-stranded DNA, thereby ligating cohesive ends of double-stranded DNA or repairing nicks in double-stranded DNA. E. coli DNA Ligase can catalyze phosphodiester bond formation between adjacent 5'-phosphate and 3'-hydroxyl groups at cohesive ends of double-stranded DNA, but cannot ligate blunt-ended double-stranded DNA under standard conditions.It can catalyze the ligation of blunt-ended double-stranded DNA in the presence of 10–15% PEG and appropriately high concentrations of monovalent cations. E. coli DNA Ligase is active over a temperature range of 4–37°C and can be heat-inactivated by incubation at 65°C for 20 minutes.
| Source | Recombinant expressed in Escherichia coli
| | Appearance | Sterile liquid
| | Storage Buffer | 10mM Tris-HCl, 50mM KCl, 1mM DTT, 0.1mM EDTA, 200µg/ml BSA, 50% Glycerol, pH7.4 @25℃.
| | Enzyme Concentration | 10 U/μl | | Purity | Free of other DNA ligases except E. coli DNA Ligase. Free of endonucleases, exonucleases, RNases and phosphatases.
| | Activity Definition | One unit is defined as the amount of enzyme required to give 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℃ in 1× E. coli DNA Ligase Reaction Buffer. |
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Component List
E745500
| Component | 200U
| 1KU | 5×1KU | Storage | | E745500A | E. coli DNA Ligase (10U/μl) | 20µl | 100µl | 5×100µl | -20℃. Avoid freeze/ Thaw cycle. | | E745500B | 10× Reaction Buffer | 100µl
| 500µl
| 5×500µl | -20℃. Avoid freeze/ Thaw cycle. |
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Product Applications
E. coli DNA Ligase catalyzes the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl groups at the cohesive ends of double-stranded DNA. It is a DNA ligase that performs ligation of cohesive-ended double-stranded DNA or nick repair of double-stranded DNA.
Product Advantages
E. coli DNA Ligase is active over a wide temperature range (4–37 °C) and can be heat-inactivated at 65 °C for 20 minutes. It shows high ligation efficiency for cohesive-ended linear double-stranded DNA.
Instructions for Use
1.Prepare the reaction mixture on ice according to the table below (using a 20 μl system as an example):
| Reagent | Volume | Final Concentration |
|---|
| dsDNA | x μl | up to 0.25 μg/μl | | 10× Reaction Buffer | 2 μl | 1× | | E. coli DNA Ligase (10U/μl) | 1 μl | 0.5U/μl | | Nuclease-free Water | (17−x) μl | — | | Total Volume | 20 μl | — |
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Note 1. If multiple reactions are performed simultaneously, all components except the substrate DNA in the table above can be pre-mixed and then aliquoted into each reaction tube.
Note 2. E. coli DNA Ligase should be kept in an ice box or on ice during use.
2. Mix gently by vortexing or pipetting up and down, then centrifuge at low speed to bring any liquid adhering to the tube walls down to the bottom.
3. Reaction conditions: Incubate at 16°C for 30–60 minutes.
4. Termination of reaction: Immediately after the reaction, incubate the product at 65°C for 20 minutes to terminate the reaction.
5. Analyze the ligation product by agarose or polyacrylamide gel electrophoresis and photograph to observe and assess ligation efficiency. If DNA samples need to be recovered from agarose gels, a DNA gel recovery kit is recommended; if DNA samples need to be purified from restriction digestion reaction systems, a PCR purification kit / DNA purification kit is recommended.
Frequently Asked Questions
1. What is the difference between E. coli DNA Ligase and T4 DNA Ligase?
Under recommended conditions, E. coli DNA Ligase cannot ligate blunt-ended DNA or RNA molecules.
2. Can E. coli DNA Ligase be heat-inactivated?
Yes. Incubating E. coli DNA Ligase at 65°C for 20 minutes will completely inactivate the enzyme.
3. Which ligase or ligation kit should be chosen?
For rapid ligation of blunt or cohesive ends, a rapid DNA ligation kit is recommended. T4 DNA Ligase is the enzyme of choice for most DNA recombination reactions, suitable for ligation of cohesive ends (10 minutes at room temperature) or blunt ends (2 hours at room temperature or overnight). E. coli DNA Ligase is more substrate-specific than T4 DNA Ligase. If only cohesive end ligation is required and blunt end or RNA ligation needs to be inhibited, E. coli DNA Ligase is recommended. For ligation of single-stranded DNA or RNA molecules, T4 RNA Ligase is recommended.
Storage Conditions
Store at -20°C with a two-year validity period. For the 10× Reaction Buffer, long-term storage at -80°C is recommended to extend the half-life of NAD.
Precautions
(1) E. coli DNA Ligase requires NAD⁺ (nicotinamide adenine dinucleotide) as a cofactor, unlike T4 DNA Ligase and others which use ATP.
(2) E. coli DNA Ligase has extremely low ligation efficiency for blunt-ended fragments; T4 DNA Ligase is recommended for blunt end ligation.
(3) E. coli DNA Ligase catalyzes reactions on double-stranded DNA molecules and cannot be used for ligation of single-stranded DNA or RNA.
(4) E. coli DNA Ligase ligation reactions require NAD as a cofactor. The 10× Reaction Buffer contains NAD, so it is recommended to store the 10× Reaction Buffer long-term at -80°C to extend the half-life of NAD.
(5) This product is for scientific research use only by qualified personnel. It is not for clinical diagnosis or treatment, not for use in food or pharmaceuticals, and must not be stored in ordinary residential premises.
(6) For your safety and health, please wear a lab coat and disposable gloves when handling.
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