T5 Exonuclease is a highly efficient 5´→3´ exonuclease for either ssDNA or dsDNA. It also has ssDNA specific endonuclease activity in the presence of magnesium ions. However, the enzyme does not degrade supercoiled dsDNA (1). The mode of action of T5 Exonuclease in vivo may be analogous to that of the 5´→3´ exonuclease activity of E. coli DNA Polymerase I (1, 2).

The physical purity of this enzyme is ≥98% as assessed by SDS-PAGE with Coomassie® blue staining (see figure below).


Product Source
E. coli strain expressing the T5 phage D15 gene.


  • Plasmid mutagenesis methods.
  • Oligonucleotide site-directed mutagenesis.
  • Generation of plasmid-sequencing templates.
  • Generation of 3’-overhang for improved cloning procedures.

Product Includes
1)   T5 Exonuclease
2)   10x T5 Exonuclease Reaction Buffer

Storage Temperature
–20 °C

Storage Buffer

50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25 ºC 

10x T5 Exonuclease Reaction Buffer

330 mM Tris-acetate (pH 7.5), 660 mM potassium acetate, 100 mM magnesium acetate, 5.0 mM DTT

Unit Definition

One unit of T5 Exonuclease catalyzes the release of 1 nmol of acid-soluble nucleotides from double-stranded calf thymus DNA in 30 minutes at 37 °C under standard assay conditions.

Quality Control

T5 Exonuclease is free from detectable RNase or contaminating DNA endonuclease activities.3372 3374