Inefficient Cleavage Due to Single Recognition Site

Symptom

Incomplete digestion persists despite adequate enzyme units and incubation time; some restriction enzymes require two recognition sites on the same DNA molecule for efficient cleavage.

1. 1 Enzyme requires presence of two recognition sites for efficient catalytic activity
2. 2 DNA substrate contains only single recognition site

1. 1 Check enzyme product information to confirm two-site requirement
2. 2 Use alternative restriction enzyme with same specificity but no two-site requirement
3. 3 Add control DNA fragment containing the recognition site to provide second site in trans
4. 4 Increase enzyme concentration and incubation time for partial activity on single sites

Related Video (1)

Addgene ★ 85

Restriction Digest Analysis

"Directly demonstrates restriction enzyme digestion and gel analysis, showing how incomplete cleavage would appear and how to assess digest efficiency."