Exonuclease I, E. coli (ExoI)

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 37ºC. 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 25°C, 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 37ºC for 15 min., 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 Stability: May 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.