Cloning With Restriction Enzymes

Restriction enzymes recognize specific DNA sequences and generate double-stranded breaks in the DNA duplex. Cleavage produces fragments with either sticky ends (5' or 3' overhangs) or blunt ends.

After restriction enzyme cleavage, the 5' phosphates and 3' hydroxyls are maintained on DNA ends, leaving them ready to be joined together via ligation. This property makes restriction enzymes valuable for recombinant DNA technologies.

Select restriction enzymes that will produce DNA ends compatible with your chosen vector. This ensures the insert fragments can be properly ligated into the cloning vector.

Confirm that your selected restriction enzyme recognition sites do not occur within the DNA fragment to be cloned. This prevents unwanted internal cuts that would fragment your insert.

Check the methylation sensitivity of your selected enzyme to ensure that host methylation by dam and/or dcm methylases will not block cleavage. This prevents failed digestion due to bacterial methylation patterns.

Use NEB's online interactive tools to aid in selecting the appropriate restriction enzyme and to determine optimal reaction parameters. Visit CLONEWITHNEB.com for a complete list of available products.