T5 Exonuclease
T5 Exonuclease
Greater than 95% as determined by SDS-PAGE.
Description
T5 Exonuclease T5 phage D15 gene Recombinant produced in E.Coli is a single, non-glycosylated polypeptide. T5 Exonuclease is purified by proprietary chromatographic techniques.
Product Specs
T5 Exonuclease, an essential enzyme in the exonuclease family, plays a critical role in DNA metabolism and genetic engineering. This research paper aims to provide a comprehensive overview of T5 Exonuclease, encompassing its structure, function, and diverse applications in molecular biology.
Derived from bacteriophage T5, T5 Exonuclease exhibits a remarkable ability to selectively degrade single-stranded DNA in a 5' to 3' direction. Its highly processive nature allows it to cleave multiple nucleotides sequentially without detaching from the DNA substrate. The enzyme's high specificity for single-stranded DNA makes it an invaluable tool for various molecular biology applications.
The primary function of T5 Exonuclease is to remove nucleotides from the 5' ends of single-stranded DNA molecules. Its processive DNA digestion capabilities make it essential for DNA repair mechanisms, such as removing damaged or mismatched nucleotides. T5 Exonuclease is also widely employed in molecular cloning techniques to generate DNA fragments with precise ends for subsequent ligation reactions.
Recombinantly produced in E. coli, T5 Exonuclease is a single, non-glycosylated polypeptide derived from the T5 phage D15 gene. Purification of T5 Exonuclease is achieved through proprietary chromatographic techniques.
The formulation consists of 10U/ul T5 Exonuclease in a buffer containing 50mM Tris-HCl (pH 7.5 at 25°C), 100mM NaCl, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, and 50% glycerol.
Avoid repeated freezing and thawing.
SDS-PAGE analysis indicates a purity greater than 95%.
One unit (1U) of T5 Exonuclease is defined as the amount of enzyme required to produce a change in absorbance at 260nm of 0.00032 per minute (0.00032 A260nm/min) at 37°C in a reaction buffer containing 20mM Tris-acetate (pH 7.9 at 25°C), 50mM Potassium Acetate, 10mM Magnesium Acetate, and 1mM DTT (1x Reaction Buffer).
One application of this product is in Gibson Assembly reactions.
T5 Exonuclease
Product Science Overview
T5 Exonuclease is a versatile enzyme derived from the bacteriophage T5. It exhibits both exonuclease and endonuclease activities, making it a valuable tool in molecular biology. The recombinant form of T5 Exonuclease is produced through the expression of the T5 phage D15 gene in Escherichia coli (E. coli), allowing for large-scale production and consistent quality.
T5 Exonuclease specifically degrades double-stranded DNA (dsDNA) from the 5’ to 3’ direction. It initiates nucleotide removal from the 5’ termini or at gaps and nicks of linear or circular dsDNA. Additionally, T5 Exonuclease has single-stranded DNA (ssDNA) endonuclease activity, enabling it to cleave ssDNA at specific sites .
T5 Exonuclease is widely used in various molecular biology applications, including:
- Gibson Assembly: This technique involves the use of T5 Exonuclease to create single-stranded DNA overhangs by chewing back the 5’ ends of dsDNA. These overhangs facilitate the annealing of complementary DNA fragments, which are then joined by DNA polymerase and ligase .
- DNA Cloning: T5 Exonuclease is employed to remove incomplete ligation products from ligated circular dsDNA, enhancing the efficiency of cloning procedures .
- Plasmid Purification: The enzyme degrades denatured DNA from alkaline-based plasmid purification methods, improving the quality of the purified plasmid DNA .
The recombinant T5 Exonuclease is produced by cloning the T5 phage D15 gene into an expression vector, which is then transformed into E. coli. The bacteria are cultured, and the enzyme is expressed as a His-tag fusion protein. The recombinant enzyme is subsequently purified using affinity chromatography techniques, ensuring high purity and activity .
T5 Exonuclease catalyzes the hydrolysis of phosphodiester bonds in DNA, resulting in the release of nucleotides. The enzyme’s activity is influenced by various factors, including temperature, pH, and the presence of divalent cations such as magnesium ions. Optimal reaction conditions for T5 Exonuclease include a temperature of 37°C and a pH of 7.9 .
