Description: Catalyzes the removal of nucleotides from single-stranded DNA in the 3' to 5' direction.
Source: An E. coli strain that carries the cloned Exo I gene from E. coli NM554.
Applications:
Exonuclease I degrades excess single-stranded primer oligonucleotide from a reaction mixture containing double-stranded extension products
Reagents Supplied: Exonuclease I Reaction Buffer (10X)
Enzyme Properties Heat Inactivation: 80°C for 20 minutes
Reaction & Storage Conditions Reaction Conditions: 1X Exonuclease I Reaction Buffer Incubate at
37°C.
1X Exonuclease I Reaction Buffer: 67 mM Glycine-KOH 6.7 mM MgCl2 10 mM 2-Mercaptoethanol
pH 9.5 @ 25°C
Unit Definition: One unit is defined as the amount of enzyme that will catalyze the release of 10 nmol of acid-soluble nucleotide in a total reaction volume of 50 μl in 30 minutes at 37°C in 1X Exonuclease I Reaction Buffer with 0.17 mg/ml single-stranded [3H]-DNA.
Concentration: 20,000 units/ml
Storage Conditions: 10 mM Tris-HCl 100 mM NaCl 5 mM 2-Mercaptoethanol 0.5 mM EDTA 100 µg/ml BSA 50% Glycerol
pH 7.5 @ 25°C
Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product.
Single-stranded Endonuclease Assay
: Incubation of 100 units of Exonuclease I with 1 µg of M13mp18 single-stranded DNA for 16 hours at 37°C in a 50 µl reaction resulted in no decrease in the amount of closed circular DNA as determined by agarose gel electrophoresis.
Double-stranded Exonuclease Assay: Incubation of 50 units of enzyme with 0.2 μg 3H DNA (9.0 x 103 cpm/μg) for 4 hours at 37°C in 50 μl reaction buffer released <0.5 % radioactivity.
Endonuclease Activity: Incubation of a 50 μl reaction containing 100 units of Exonuclease I (E. coli) with 1 μg of ΦX174 RF I DNA for 16 hours at 37ºC resulted in < 5% conversion to RFII as determined by agarose gel electrophoresis.
References
Lehman and Nussbaum (1964) J. Biol. Chem., 239, 2628.
Kusher, et al. (1971) Proc. Natl. Acad. Sci. USA, 68, 824.
Kusher et al. (1972) Proc. Natl. Acad. Sci. USA, 69, 1366.
Goldmark and Linn (1972) J. Biol. Chem., 247, 184.
