In restriction digest validation, mismatch indicates?

• A. An incorrect plasmid construct has been prepared.
• B. The gel ran incorrectly.
• C. The enzyme did not cut.
• D. The DNA sample is degraded.

Answer: (A)

Restriction digest validation relies on comparing the observed fragment pattern to the expected pattern for the intended plasmid. When there is a mismatch, it signals that the plasmid you prepared does not match what you designed, often due to the wrong insert, incorrect orientation, or unintended sequence changes that alter fragment sizes. This interpretation is distinct from issues like the gel running incorrectly (which would affect many lanes or appear as odd lane quality), the enzyme not cutting (which would leave mostly undigested, larger bands), or DNA degradation (which would produce smeared or smeared-like patterns). The fingerprint should line up with the planned construct, and a mismatch points to an incorrect plasmid design or assembly.