One unit is defined as the amount of enzyme required to cleave 50% of 0.2 pmol of a single T:T mismatch in 30 minutes at 37°C in a total reaction volume of 20 µl in 1X NEBuffer r2.1. 1 μl of Mismatch Endonclease I contains 80 U, therefore, 1 µl of Mismatch Endonuclease I is able to completely cleave 8 pmol of mismatched DNA.