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 (2)

Addgene ★ 80

Restriction Digest Analysis

"Shows actual digestion and gel analysis, directly relevant to troubleshooting incomplete cleavage."

New England Biolabs ★ 75

Cloning With Restriction Enzymes

"Explains restriction enzyme selection and usage, covering the need for two sites."