Product Description:
Exonuclease I cleaves single-stranded DNA in the 3′→ 5′ direction, releasing 5′ -mono/di-nucleotides and leaving double-stranded DNA molecules and the 5'-terminus intact. The enzyme is processive though digestion is inhibited by the presence of a 3′ -terminal phosphate. Exonuclease I is tolerant of a wide-range of buffer conditions and can typically be added to reactions containing magnesium (1-3).

Source of Protein
Purified from a strain of E. coli that expresses the recombinant Exonuclease I gene.

Supplied in
10 mM Tris-HCl
100 mM NaCl
1 mM dithiothreitol
0.5 mM EDTA
50% glycerol
pH 7.5 @ 25°C

Unit Definition
One unit is defined as the amount of enzyme required to produce 10 nmol of acid-soluble total nucleotide in 30 minutes at 37°C.

Nuclease Contamination Tests:
Endonuclease Activity
A 50 µL reaction containing 1 µg of pBR322 DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.

E.coli 16S rDNA Contamination Test
Replicate 5 µL samples of enzyme solution were heat denatured and screened in a TaqMan qPCR assay for the presence of contaminating E.coli genomic DNA using primers for the 16S rRNA locus. Based on no template control C_t values, the detection limit of this assay is <10 copies genome/sample.

Notes:

1. Exonuclease I can be heat-inactivated by incubation at 80°C for 15 minutes.
2. Exonuclease I will preferentially degrade single-stranded oligonucleotide primers in a reaction containing amplification products or other sources of double-stranded DNA, leaving double-stranded molecules intact.
3. Exonuclease I, in combination with Lambda Exonuclease (X803L), is effective in removing linear DNA species from plasmid preparations.
4. Exonuclease I works well in most molecular biology buffers which contain magnesium in excess of 1.5 mM.