NEBcutter 2.0 was New England Biolabs’ classic tool for finding restriction enzyme sites in a DNA sequence. It has since been succeeded by NEBcutter V3.0, and the old 2.0 address now redirects to the current version. The official tool is embedded below so you can run your analysis here, exactly as before.
Open NEBcutter in a new tab ↗ | Go to the NEBcutter V3.0 guide →
NEBcutter 2.0 vs 3.0: what changed
If your protocol or bookmark references NEBcutter 2.0, you can keep using it — the analysis is the same, now running on the updated V3.0 engine. V3.0 adds a refreshed enzyme database, a faster interface and improved handling of larger sequences. There is no separate 2.0 server to maintain any more; NEB consolidated everything into V3.0.
How to run a restriction analysis
- Enter your DNA (raw sequence, FASTA, or a GenBank/EMBL accession) and choose linear or circular topology.
- Select the enzyme set — all enzymes, NEB-supplied only, or a custom list.
- Review the map of cut sites, single cutters, non-cutters and predicted fragment sizes for your gel.
What it’s used for
- Cloning — find single cutters to linearise a vector or excise an insert.
- Diagnostic digests — predict the band pattern that confirms a construct.
- RFLP and mapping — compare cut patterns between sequences.
For a full walkthrough of reading the output, choosing enzymes (single cutters, methylation sensitivity, double-digest buffers) and interpreting fragment sizes, see the complete NEBcutter V3.0 guide.
Frequently asked questions
Does NEBcutter 2.0 still work?
The 2.0 address now redirects to NEBcutter V3.0, which runs the same analysis. The tool embedded on this page is the current version.
Is it free?
Yes, NEBcutter is free from New England Biolabs and runs in the browser with no installation.
Should I use 2.0 or 3.0?
Use V3.0 — it is the current, maintained version. Results are equivalent to the old 2.0 for standard analyses.
Related guide: Restriction enzyme cloning: a step-by-step guide →
