T4 DNA
T4 DNA Ligase Recombinant
Cat. No.
BT5080
Source
Escherichia Colilambda lysogen NM 989.
Synonyms
DNA ligase 4, EC 6.5.1.1, DNA ligase IV, Polydeoxyribonucleotide synthase [ATP] 4.
Appearance
Sterile filtered liquid formulation having a concentration of 167,000 U/ml.
Purity
Usage
Prospec's products are furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.
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Description
T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5' -phosphate and 3' -hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.
Product Specs
Description
T4 DNA Ligase is an enzyme that facilitates the creation of a phosphodiester bond between the 5' phosphate and 3' hydroxyl termini of adjacent DNA or RNA strands. It can join both blunt and cohesive ends, and repair single-strand nicks in duplex DNA, RNA, or DNA/RNA hybrids.
Physical Appearance
A liquid solution, sterilized by filtration, with a concentration of 167,000 units per milliliter.
Formulation
The formulation contains 50mM Tris-HCl (pH 7.8 at 25 degrees Celsius), 10mM MgCl2, 10mM DTT, 1mM ATP, 25 micrograms/ml BSA, and DNA at a concentration of 0.1 to 1 micromolar in 5' termini. The optimal temperature for ligation is 16 degrees Celsius.
Unit Definition
Two definitions apply: 1. One unit of the enzyme is the quantity needed to ligate 50% of Hind III DNA fragments (with a 5' DNA termini concentration of 0.12 micromolar, 300 micrograms/ml) in a 20 microliter reaction volume within 30 minutes at 16 degrees Celsius using 1X T4 DNA Ligase Reaction Buffer. 2. One Weiss unit is defined as the enzyme amount needed to catalyze the exchange of 1 nanomole of 32P from pyrophosphate to ATP, becoming Norit-adsorbable, within 20 minutes at 37 degrees Celsius.
Biological Activity
One Weiss unit is roughly equivalent to 67 cohesive-end ligation units. Notably: T4 DNA Ligase is significantly inhibited by NaCl or KCl concentrations exceeding 200mM. Ligation of blunt-ended and single-base pair overhang fragments requires approximately 50 times more enzyme compared to cohesive-end DNA fragments to achieve the same ligation extent. Enhancing blunt-end ligation can be achieved by adding PEG 4000 (10% w/v final concentration), hexamine chloride, or lowering the ATP concentration to 50 micromolar. For storing diluted T4 DNA Ligase at -20 degrees Celsius, use a 50% glycerol storage buffer; for immediate use, dilute with 1x T4 DNA Ligase reaction buffer.
Inactivation
To inactivate T4 DNA Ligase, incubate it at 65 degrees Celsius for 10 minutes.
Note
This product is purified to remove endonucleases and exonucleases. Each batch undergoes a mock cloning assay to detect any damage to ligated DNA termini. This assay consistently shows that over 99.9% of the termini remain undamaged.
Exonuclease Activity
Less than 0.3% of total radioactivity was released when 1 microgram of a single and double-stranded [3H] E. coli DNA mixture (200,000 cpm/ug) was incubated with 13,000 units of T4 DNA Ligase in a 50 microliter reaction for 4 hours at 37 degrees Celsius.
Endonuclease Activity
Incubating 1 microgram of X174 RF I DNA with 13,000 units of T4 DNA Ligase in a 50 microliter reaction for 4 hours at 37 degrees Celsius resulted in less than 5% conversion to RFII, as determined by agarose gel electrophoresis.
Nuclease Activity
A clear and distinct banding pattern was observed on agarose gels after incubating 13,000 units of T4 DNA Ligase in assay buffer (excluding ATP) with Hind III fragments of gamma DNA for 18 hours.
Applications
This enzyme is used for cloning restriction fragments and joining linkers and adapters to DNA with blunt ends.
Synonyms
DNA ligase 4, EC 6.5.1.1, DNA ligase IV, Polydeoxyribonucleotide synthase [ATP] 4.
Source
Escherichia Colilambda lysogen NM 989.
Storage Buffer
50mM KCl, 10mM Tris-HCl (pH 7.4), 0.1mM EDTA, 1mM DTT, 200 µg/ml BSA and 50% glycerol. Store at -20C.
Product Science Overview
Function and Mechanism
T4 DNA Ligase is responsible for catalyzing the formation of phosphodiester bonds between adjacent nucleotides in DNA. This process is essential for repairing breaks or “nicks” in the DNA backbone that can occur during DNA replication, recombination, and repair. The enzyme works by joining the 3’-hydroxyl (OH) end of one nucleotide with the 5’-phosphate (P) end of another, effectively sealing the gap and restoring the integrity of the DNA molecule .
The mechanism of T4 DNA Ligase involves three main steps :
- Enzyme Adenylation: The enzyme first binds to a molecule of adenosine triphosphate (ATP) and transfers an adenosine monophosphate (AMP) group to itself.
- AMP Transfer: The AMP group is then transferred to the 5’-phosphate end of the DNA at the site of the nick.
- Phosphodiester Bond Formation: The 3’-hydroxyl group of the adjacent nucleotide attacks the 5’-phosphate-AMP complex, resulting in the formation of a phosphodiester bond and the release of AMP.
Applications in Molecular Biology
T4 DNA Ligase is widely used in various molecular biology applications, including :
- Cloning: The enzyme is used to ligate DNA fragments into vectors, such as plasmids, to create recombinant DNA molecules. This is a critical step in the cloning process, allowing for the insertion of genes or other DNA sequences into vectors for propagation and expression in host cells.
- DNA Repair: T4 DNA Ligase is employed in DNA repair assays to study the mechanisms of DNA damage and repair.
- Library Construction: The enzyme is used in the construction of genomic and cDNA libraries, where it helps to ligate DNA fragments into vectors for sequencing and analysis.
- Site-Directed Mutagenesis: T4 DNA Ligase is used to introduce specific mutations into DNA sequences by ligating oligonucleotides containing the desired mutations into target DNA.
Advantages of Recombinant T4 DNA Ligase
The recombinant form of T4 DNA Ligase offers several advantages over the native enzyme :
- High Purity: Recombinant production allows for the purification of the enzyme to a high degree of purity, reducing the risk of contamination with other proteins or nucleases.
- Consistency: Recombinant production ensures a consistent and reliable supply of the enzyme, with uniform activity and performance across different batches.
- Cost-Effectiveness: Large-scale production of recombinant T4 DNA Ligase is more cost-effective than isolating the enzyme from natural sources, making it more accessible for research and industrial applications.
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