Virtual Restriction Digest and Gel Fragment Simulator
Digest the cited pUC19 reference with EcoRI and HindIII and inspect the predicted fragment sizes. The calculation uses the actual circular sequence, enzyme definitions and methylation setting. Its gel view is a schematic aid to interpreting fragment sizes, not a calibrated prediction of migration in a particular gel system.
Versioned pUC19 sequence with two selected restriction enzymes and the example’s stated dam/dcm host setting. The map, fragment calculation and schematic gel derive from the same sequence and enzyme table. No wet-lab gel image is used as synthetic evidence.
Check recognition sites and cut coordinates separately
Restriction enzymes recognise a sequence but can cut at offsets from it. The engine therefore needs both recognition and cleavage definitions. This distinction is especially important for Type IIS enzymes, but ordinary double digests also require the correct strand and cut positions. A text search for a recognition motif alone is not a complete fragment calculation.
The pUC19 reference provides a compact example where the selected sites lie in a familiar cloning region. Follow each site on the map and compare the ordered cut positions with the fragment table. The sum of complete-digest fragment lengths should equal the input sequence length. That conservation check catches interpretation mistakes without relying on the appearance of the gel drawing.
Use a simulated digest to plan an informative experiment
A useful diagnostic digest distinguishes plausible constructs, such as an intended insert, an empty vector or a reversed insert. Merely producing two bands is not enough if every candidate produces the same sizes. Consider the expected fragment separation and whether the smallest informative fragment is likely to be visible under the planned gel conditions.
Retain the enzyme set, host methylation assumption and input sequence with the prediction. If an experimental gel disagrees, consider partial digestion, mixed plasmid populations, topology and sequence differences before concluding that the calculation was wrong. The simulation describes the stated complete-digest model. Sequencing or another orthogonal check may be needed when several physical explanations produce similar band patterns.
Equations and variables
Fragment lengths between ordered cuts
li = cuti+1 − cuti; lwrap = L − cutlast + cutfirst
- li
- Length between neighbouring cuts (bp)
- L
- Circular molecule length (bp)
- lwrap
- Fragment that crosses the coordinate origin (bp)
Length conservation
Σ li = L
- li
- Lengths of all predicted complete-digest fragments (bp)
- L
- Input molecule length (bp)
Scope and limitations
- The digest assumes the stated sequence and applicable enzyme cuts. Partial digestion, star activity, damaged DNA and reaction inhibition are not inferred.
- Methylation can prevent or qualify a cut. Unknown sensitivity must remain explicit rather than being silently treated as an assured cleavage.
- Similar-sized fragments may be unresolved experimentally, and very small fragments may be difficult to detect. A schematic band is not a guarantee of visibility.
Sources and provenance
- GenBank pUC19 record L09137.2Existing source notice: NCBI public domain
Versioned sequence and SHA-256 are pinned in the shipped example.
- Restriction enzyme information in REBASEReference link; shipped enzyme table retains its own source notices
Technical questions
Why can a circular molecule give a different fragment count from a linear one?
A circular molecule cut at k distinct positions generally produces k fragments, while a linear molecule with k internal cuts generally produces k + 1. The uncut ends of a linear molecule matter. Confirm topology before treating a fragment-count difference as an engine error.
Does the gel image predict exact migration distance?
No. The schematic communicates fragment-size relationships. Real migration depends on gel concentration, buffer, voltage, DNA topology and other conditions. Use an appropriate size ladder and experimental controls when interpreting an actual gel.
What if two fragments have almost the same size?
Their predicted lengths remain distinct even when a real gel may show one unresolved band. Inspect the fragment table and expected resolution rather than counting only drawn bands. A single experimental band can contain more than one fragment.
Connected workflows
- GenBank and FASTA plasmid mapping
Inspect the underlying sequence and site locations.
- Type IIS restriction-site inspection
Review cleavage outside Type IIS recognition sites.
- Vector and insert molar-ratio calculation
Plan amount and identity checks for an assembled construct.
- Dam and Dcm Methylation Restriction Block Checker (pUC19)
See which digest sites a dam+/dcm+ host methylates and blocks.