Restriction Digest Calculator
Calculate expected fragment sizes from restriction enzyme digest of known DNA.
Linear Versus Circular DNA
The number of fragments produced depends on the topology of the starting molecule, and this catches people out constantly.
| DNA form | n cut sites | Fragments |
|---|---|---|
| Linear | n | n + 1 |
| Circular | n | n |
| Circular | 1 | 1 — linearised, not cut into pieces |
A plasmid cut once becomes a single linear molecule of the full length. Cut three times, it yields three fragments — not four — because the ends join around the circle. For linear DNA the same three cuts give four pieces.
Fragment sizes for a circular molecule are the differences between consecutive cut positions, with the final fragment wrapping from the last site back to the first.
Reading a Gel
| Observation | Likely cause |
|---|---|
| Fragment count wrong by one | Linear versus circular confusion |
| Fragments of identical size | Co-migrate — appear as one brighter band |
| Very small fragments missing | Run off the gel or too faint to stain |
| Extra high band | Incomplete digestion — partially cut molecules |
| Smearing | Star activity or degraded DNA |
Band intensity is proportional to mass, not molar amount. A 4 kb fragment stains four times as brightly as a 1 kb fragment present in the same molar quantity, which is why small fragments are easy to miss entirely.
Star activity — cleavage at non-canonical sites — occurs with excessive enzyme, prolonged incubation, glycerol above about 5%, or the wrong buffer. It produces unexpected extra bands and is the usual explanation when a digest gives more fragments than the map predicts.
Worked Examples
Common Mistakes
Circular DNA gives n fragments from n cuts, not n+1. A singly cut plasmid is linearised rather than fragmented.
Two fragments of similar size run as one band, appearing brighter. Fragment counts from a gel can therefore understate the true number.
Staining intensity scales with mass, so short fragments are faint and may run off the gel entirely. Adjust gel percentage and run time accordingly.
Too much enzyme, long incubation or high glycerol causes cleavage at non-canonical sites, producing extra unexpected bands.