FAQ: How is Thermolabile USER® II Enzyme different than USER Enzyme?

Thermolabile USER II Enzyme is similar to USER (Uracil-Specific Excision Reagent) Enzyme (NEB #M5505), in that both will generate a single nucleotide gap at the location of a uracil, with the added benefit of being fully heat inactivated at 65°C. The lyase activity of Endonuclease VIII in USER Enzyme breaks the phosphodiester backbone and leaves a 3´ and 5´ phosphate, whereas the lyase activity of Endonuclease III in Thermolabile USER II Enzyme breaks the phosphodiedester backbone and leaves a 3'-phospho-α, β-unsaturated aldehyde and a 5' phosphate.