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Restriction Digest Planner

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.

Molecular BiologyREBASE Database250+ EnzymesClient-Side

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Load example restriction digest data to see the full workflow

DNA Sequence

0 bp

Select Enzymes

  • Plan single, double, or multi-enzyme restriction digests
  • Check methylation sensitivity for Dam/Dcm/CpG before digesting
  • Assess star activity risk and find HF enzyme alternatives
  • Find optimal shared buffer for double digests
  • Predict fragment sizes and simulate agarose gel patterns
  • Visualize cut sites on linear or circular sequence maps

Don't use for

  • Golden Gate or Gibson assembly — these use Type IIS enzymes with different logic
  • Very long genomic sequences (>100 kb) — the client-side parser may be slow
  • Custom methylation patterns — the tool flags methylation sensitivity but does not model specific methylation states of your DNA

Restriction Enzymes in Molecular Cloning

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.

Methylation Sensitivity and Star Activity

Dam methylation (GATC) and Dcm methylation (CCWGG) are common in E. coli-grown DNA. Enzymes like MboI and ClaI are blocked by Dam; BstNI by Dcm. CpG methylation in mammalian DNA blocks enzymes like HpaII and SmaI.
Star activity is nonspecific cleavage at altered sequences under suboptimal conditions. Risk factors include: • High enzyme:DNA ratio (>10 units/µg) • Extended incubation (>4 hours) • High glycerol concentration (>5% v/v) • Low ionic strength or non-optimal pH

NEB High-Fidelity (HF) enzyme variants are engineered to minimize star activity. When available, HF versions are preferable for overnight digests.

Agarose Gel Electrophoresis

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.

Frequently Asked Questions

Next Steps

Gel Electrophoresis Systems

Horizontal gel boxes and power supplies for DNA analysis

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Gel Documentation Systems

UV and blue-light gel imaging systems

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