Invitrogen™ Exonuclease I

Product Code.	Quantity	unitSize
15513677	2500 U	2500 units

Product Code. 15513677 Supplier Invitrogen™ Supplier No. 70073Z2500UN

Description

Useful in preparing PCR products for applications involving sequencing or labeling methods

Exonuclease I is particularly useful in preparing the products of PCR for applications involving sequencing or labeling methods¹. Typically, the excess primers and any other extraneous single-stranded DNA present in PCR products will interfere with subsequent enzymatic reactions involving DNA synthesis. The hydrolytic properties of Exonuclease I degrade all single-stranded DNA present in the PCR mixture allowing the product to be used more efficiently in other applications.

• Hydrolyzes single-stranded DNA in the 3′-5′ direction, releasing 5′-mononucleotides and leaving the terminal 5′-dinucleotide intact; hydrolysis is processive and cannot proceed if the 3′ terminus is phosphorylated
• Use to measure the endonucleolytic cleavage of covalently closed circular single-stranded DNA reacted with an endonuclease of interest²
• Also suitable for measuring DNA helicase activity³
• When combined with Shrimp Alkaline Phosphatase (Mfr. No. 78390) for dNTP dephosphorylation, the use of alternative purification methods, such as columns, gels or magnetic separations, are completely eliminated

Source: E. coli strain containing an overproducing clone of E. coli Exonuclease I
Properties:

• Molecular Weight: 55 kDa
• Heat Inactivation: 80° for 15 min.
• Degrades to terminal dinucleotides
• Degrades glycosylated DNA
• Optimum Temperature: 37°C

Purity:

• Greater than 95% pure as determined by SDS-PAGE
• Tested for contaminating endonucleases, double-stranded exonucleases and ribonucleases

Storage Buffer:
20mM Tris-HCI (pH 7.5), 5mM 2-mercaptoethanol, 0.5mM EDTA, 50% glycerol
Assay Conditions:

Reaction mixture (100μL) contains 67mM glycine buffer (pH 9.5), 10mM
2-mercaptoethanol, 6.7mM MgCl₂, 0.5mM denatured DNA and enzyme

Incubation is at 37°C for 30 min.

Unit Definition: One unit is the amount of enzyme which catalyzes the release of 10nmol of acid-soluble nucleotide from denatured DNA in 30 min at 37°C under standard conditions
Concentration:

• Standard Conc.: 10 units/μL, Mfr. No. 70073
• High Conc.: 20 units/μL, Mfr. No. 72073

Functional Assay: Treated PCR product with Exonuclease I to degrade unincorporated primers before performing sequencing reaction with Sequenase™ Version 2.0 DNA Polymerase Sequencing Kit (Mfr. No. 70770)

Elimination of residual single-stranded DNA containing a 3′ terminus; Measuring endonucleolytic cleavage of covalently closed circular ssDNA; Measuring DNA helicase activity

Specifications

• Concentration: 10 U/μL
• Quantity: 2500 U
• Product Type: Exonuclease I