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.

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 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 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:
Inhibitors: 20%(w/v) PEG 8000 (5)
Inactivated by heating at 80°C for 15 min.

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.