Applied Biosystems™ Exonuclease I, standard concentration, (10 units/μL)

Description

• Description: Exonuclease I 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
• Exonuclease I can be used to measure the endonucleolytic cleavage of covalently closed circular single-stranded DNA reacted with an endonuclease of interest
• In addition, DNA helicase activity can be measured utilizing Exonuclease I
• Exonuclease I is particularly useful in preparing the products of PCR for applications involving sequencing or labeling methods
• 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
• When combined with Shrimp Alkaline Phosphatase (PN 78390) for dNTP dephosphorylation, the use of alternative purification methods, such as columns, gels or magnetic separations, are completely eliminated
• For PCR clean up with Exonuclease I, see the USB ExoSAP-IT protocol
• The purchase of ExoSAP-IT provides a license to the methods of PCR clean up using Exonuclease I and SAP
• Properties: Molecular Weight: 55 kDa Heat Inactivation: 80°C 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), 5 mM 2-mercaptoethanol, 0.5 mM EDTA, 50% glycerol
• Assay Conditions: The reaction mixture (100 μL) contains 67mM glycine buffer (pH 9.5), 10 mM 2-mercaptoethanol, 6.7 mM MgCl 2 , 0.5 mM 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 10 nmol of acid-soluble nucleotide from denatured DNA in 30 min at 37°C under standard conditions
• Concentration: Standard Conc.: 10 units/μL, PN 70073 High Conc.: 20 units/μL, PN 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 (PN 70170)
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