Which enzymes cut my insert and vector where I need them to?

Plan a restriction digest

Choosing enzyme pairs for a clone, and the compatibility questions that decide it.

The requirement is narrower than it looks: you need enzymes that cut the polylinker where you want them, do not cut inside your insert, and work in a shared buffer at a shared temperature. Start by mapping every site in both the insert and the vector — the sites you must avoid are as important as the ones you want.

Prefer two different enzymes producing incompatible ends. Directional cloning stops the insert going in backwards and stops the vector closing on itself, which together account for most of the white colonies on a bad plate. If a single enzyme is unavoidable, dephosphorylate the vector.

Two practical constraints are easy to miss. Enzymes need bases either side of the site to bind, so a site placed at the very end of a PCR product may not be cut at all — suppliers publish how much overhang each enzyme needs. And some sites are blocked by Dam or Dcm methylation when the DNA came out of a standard laboratory E. coli strain, which is why a digest that should work sometimes simply does not.

Star activity — cutting at near-miss sites — appears with too much enzyme, too much glycerol or too long an incubation. If a digest gives more bands than the map predicts, suspect the reaction before the map.

What to use