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. Source: E. coli cells with a cloned E. coli sbcB gene. Concentration: ~20u/ul Unit Definition: One unit of E9007-85C catalyzes the release of 10 nano-moles of acid soluble nucleotides in 30 min at 37C. Activity Assay: 67mM glycine-KOH pH 9.5, 6.7mM MgCl2, 1mM DTT, 0.17mg/ml single stranded [3H]-DNA. Form: Supplied as a liquid in 20mM Tris-HCl, pH 7.5, 0.1mM EDTA, 1mM DTT and 50% glycerol. Supplied with: E9007-85C1: 10X Reaction Buffer: Supplied as a liquid in 670mM glycine-KOH, pH 9.5 at 25C, 67mM MgCl2, 10mM DTT. Applications: Suitable 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 mixtures: 1. Remove a 5ul aliquot from a reaction mixture. 2. Add 10u of E9007-85C and 1u of Shrimp Alkaline Phosphatase. 3. Mix and incubate at 37C for 15 min. 4. Inactivate the enzymes by heating at 85C 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. 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 37C. 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 37C. Double-stranded Exodeoxyribonuclease Assay: No 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 37C. 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 37C. Inhibition and Inactivation: Inhibitors: 20%(w/v) PEG 8000 (5) Inactivated by heating at 80C for 15 min. Storage and Stability: May be stored at 4C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20C. 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 Weight:
54.5
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