Plan single and double digests with 250+ restriction enzymes. Methylation sensitivity warnings, star activity assessment, buffer compatibility, gel simulation, and annotated sequence maps. Data never leaves your browser.
Try it out
Load example restriction digest data to see the full workflow
When to use
Do not use for
A diagnostic digest is only useful if you know the expected pattern for both positive and negative clones. Always run the digest on the empty vector as a control.
High-Fidelity enzyme variants (BamHI-HF, EcoRI-HF, etc.) have virtually no star activity. If you’re digesting overnight or using high enzyme concentrations, switch to the HF version.
If two enzymes have poor buffer compatibility (<50% activity in any shared buffer), digest with the first enzyme, column-purify or heat-inactivate, then digest with the second enzyme in its optimal buffer.
Most lab E. coli strains (DH5α, TOP10, BL21) are dam+/dcm+. If your DNA was grown in these strains, check the Warnings tab for methylation-sensitive enzymes. PCR products are not methylated.
Small fragments from frequent cutters (4-bp recognition) migrate very fast on standard gels. Use higher percentage agarose (2-3%) or polyacrylamide gels to resolve small fragments.
250+ enzymes from REBASE with methylation sensitivity (Dam, Dcm, CpG), star activity risk levels, and optimal buffer data from NEB technical resources. IUPAC degenerate base matching for recognition sites. Fragment sizes from cut position arithmetic (linear and circular). Gel migration by log10(size). Buffer compatibility scored across five NEB buffers.
Last validated 2026-04-07. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.
ConductScience Restriction Digest Planner (v2.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/restriction-digest-planner
Roberts RJ et al. REBASE—a database for DNA restriction and modification. Nucleic Acids Res. 2023. doi:10.1093/nar/gkac975
Restriction endonucleases (RE) are bacterial enzymes that cut DNA at specific recognition sequences. Type II restriction enzymes cut at fixed positions within or near their recognition site, making them invaluable for:
• Subcloning: Cutting insert and vector at compatible sites • Diagnostic digests: Verifying construct identity by fragment pattern • Restriction mapping: Determining the order of sites in unknown DNA • Genotyping: RFLP analysis for genetic variants
Enzymes produce either blunt ends (EcoRV, SmaI), 5′ overhangs (EcoRI, BamHI), or 3′ overhangs (PstI, KpnI). Compatible overhangs can be ligated; incompatible ends require blunting or adapter ligation.
NEB High-Fidelity (HF) enzyme variants are engineered to minimize star activity. When available, HF versions are preferable for overnight digests.
DNA fragments are separated by size in an agarose gel matrix under electric field. Migration distance is inversely proportional to log10(fragment size).
Gel percentage determines resolution range: • 0.5% agarose: 1-30 kb • 1.0% agarose: 0.5-10 kb • 1.5% agarose: 0.2-3 kb • 2.0% agarose: 0.1-2 kb
The simulated gel in this tool approximates a 1% agarose gel.
