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You are here:Home » Molecular Biology » Cloning-Enzymes » Exonuclease I, E. coli (ExoI)

Exonuclease I, E. coli (ExoI)

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Specifications

The Exonuclease I (ExoI) degrades single-stranded DNA in a 3'=>5' direction, releasing deoxyribonucleoside 5'-monophosphates in a stepwise manner and leaving 5'-terminal dinucleotides intact. It does not cleave DNA strands with terminal 3'-OH groups blocked by phosphoryl or acetyl groups (1).
Catalog #E9007-85C
SourceE. coli cells with a cloned E. coli sbcB gene.
Concentration ~20u/ul
Unit DefinitionOne unit of E9007-85C catalyzes the release of 10 nano-moles of acid soluble nucleotides in 30 min at 37ºC.
Activity Assay67mM glycine-KOH pH 9.5, 6.7mM MgCl2, 1mM DTT, 0.17mg/ml single stranded [3H]-DNA.
FormSupplied as a liquid in 20mM Tris-HCl, pH 7.5, 0.1mM EDTA, 1mM DTT and 50% glycerol.
Supplied withE9007-85C1: 10X Reaction Buffer: Supplied as a liquid in 670mM glycine-KOH, pH 9.5 at 25°C, 67mM MgCl2, 10mM DTT.
ApplicationsSuitable for use in the purification of PCR products, removal of single-stranded DNA containing a 3'-hydroxyl terminus from nucleic acid mixtures and assay for the presence of single-stranded DNA with a 3'-hydroxyl terminus. Other applications not tested.
Protocol for Elimination of Oligonucleotides and dNTPs from PCT mixtures1. Remove a 5ul aliquot from a reaction mixture.
2. Add 10u of E9007-85C and 1u of Shrimp Alkaline Phosphatase, P4071-05.
3. Mix and incubate at 37ºC for 15 mion.
4. Inactivate the enzymes by heating at 85ºC for 15 min.
*Note: After inactivation, the aliquot can be used directly for DNa sequencing. The Exonuclease I is not suitable for removing 3-overhangs of dsDNA.
Storage and StabilityMay be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Molecular Weight54.5kD
Quality Control
Double-stranded Endodeoxyribonuclease Assay
No detectable conversion of covalently closed circular DNA to nicked DNA was observed after incubation of 100u of Exonuclease 1 with 1ug of pUC19 DNA for 16 hours at 37°C.
Single-stranded Endodeoxyribonuclease Assay
No decrease in the amount of closed circular DNA was observed after incubation of 100u of Exonuclease 1 with 1ug of M13mp19 single-stranded DNA for 16 hours at 37°C.
Double-stranded Exodeoxyribonuclease AssayNo detectable degradation of pUC19 DNA/Alul fragments was observed after incubation of 25 units of Exonulease 1 with 1ug of digested DNA for 4 hours at 37ºC.
Ribonuclease Assay
No contaminating Ribonuclease activity was detected after incubation of 50 units of Exonuclease 1 with 1ug of [3H]-RNA for 16 hours at 37°C.
Inhibition and Inactivation
Inhibitors20%(w/v) PEG 8000 (5)
Inactivated by heating at 80°C for 15 min.
Important NoteThis product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
Alternate namesSupplied with 10X Reaction Buffer


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