When is reaction setup on ice useful in restriction digest?

Correct answer: To maintain enzyme stability and prevent premature activity before incubation

Restriction enzymes are temperature-sensitive. Preparing the reaction mixture on ice keeps the enzyme stable and limits its activity until the mixture is placed at the required incubation temperature, preventing premature DNA cutting. DNA migration in an agarose gel depends on electrophoresis conditions, not on setting up the digest on ice. Exam tip: Add the restriction enzyme last and mix the tube gently.