Restriction–ligation cloning is still the workhorse for moving a fragment into a vector. This guide covers the whole workflow — choosing enzymes, designing primers, digesting, ligating at the right ratio, and verifying — with the tools you need built into the page.
1. Plan the cut sites
Pick two restriction sites in the vector’s multiple cloning site that flank where your insert should go, ideally different enzymes for directional cloning. Crucially, confirm those sites do not cut inside your insert — check both sequences with NEBcutter. Prefer single cutters.
2. Design primers with the sites added
Amplify your insert with primers that carry the restriction site at the 5′ end, plus a few protective bases outside the site (many enzymes cut poorly at the very end of a fragment — check NEB’s cleavage-near-end data). Design the rest of the primer following our PCR primer guide; build the reverse primer with the reverse-complement tool.
3. Digest vector and insert
Double-digest both the PCR product and the vector with your two enzymes (matched buffer). Gel-purify to remove the small fragments and uncut material.
4. (Optional) Dephosphorylate the vector
If you used a single enzyme or expect high self-ligation, treat the vector with a phosphatase to reduce background re-circularisation.
5. Set up the ligation at the right molar ratio
Use a 3:1 insert:vector molar ratio as a starting point. Because longer fragments weigh more per molecule, convert the ratio to a mass with the calculator below:
Insert mass = ratio × vector mass × (insert length / vector length). A 3:1 insert:vector ratio is a common starting point for sticky-end ligations.
Always include a vector-only control to measure background.
6. Transform and verify
Transform competent cells, plate on selective medium, and screen colonies (colony PCR or miniprep + diagnostic digest). Predict the diagnostic fragment sizes with NEBcutter, and map the finished construct with PlasMapper. Confirm by sequencing.
Frequently asked questions
Why add extra bases beyond the restriction site on a primer?
Many enzymes cut inefficiently right at the end of a DNA fragment, so a few protective bases outside the recognition site improve digestion of the PCR product.
What insert:vector ratio should I use?
3:1 is a common starting point for sticky-end ligations; blunt ends often need 5:1 or more. Use the calculator above to convert to mass.
How do I avoid empty (self-ligated) vector?
Use two different enzymes for directional cloning, dephosphorylate the vector if needed, and run a vector-only control.
How do I confirm the clone?
Diagnostic restriction digest (compare to NEBcutter-predicted sizes) and sequencing; map the construct with PlasMapper.
