Restriction Fragment Calculator
This calculator determines the sizes of DNA fragments produced when restriction enzymes cut a linear or circular DNA molecule. Enter the total length of the DNA (in bp or kb) and the cut positions separated by commas. For linear DNA, the calculator includes positions 0 and the total length as boundaries. For circular DNA (e.g., a plasmid), there is no start/end boundary; the calculator automatically handles this mode. Fragments are listed from smallest to largest, mirroring a gel electrophoresis pattern.
Restriction fragment size formula
Sort cut positions + boundaries (0 and total length for linear).
Fragment size(i) = sorted position(i+1) - sorted position(i)
For circular DNA: wrap the last fragment from last cut to first cut around total length.
The sum of all fragment sizes always equals the total DNA length. This is a useful verification check after calculation.
Interpreting restriction digests
- Fragments larger than approximately 20,000 bp run slowly and appear as faint high-molecular-weight bands on standard 1% agarose gels.
- Fragments smaller than approximately 100 bp may run off a standard gel; use 2-3% agarose or polyacrylamide gels for small fragments.
- Two fragments of equal size appear as a single brighter band (double intensity) on a gel.
- Use a DNA ladder (e.g., 1 kb ladder) as a reference for accurate size estimation.
Frequently asked questions
What is a restriction enzyme?
Restriction enzymes (restriction endonucleases) are bacterial enzymes that cleave double-stranded DNA at or near specific recognition sequences. They are essential tools in molecular biology for cloning, genetic mapping, and RFLP analysis. For example, EcoRI recognizes GAATTC and cuts between G and A.
How are restriction fragment sizes calculated?
Sort the cut positions in ascending order and add the sequence boundaries (0 and total length). Fragment sizes are the differences between consecutive sorted positions: fragment 1 = position[1] - position[0], fragment 2 = position[2] - position[1], and so on. The sum of all fragments equals the total DNA length.
What is a restriction map?
A restriction map shows the positions of restriction sites along a DNA molecule. By digesting with one or more enzymes and running fragments on an agarose gel, researchers can confirm plasmid identity, verify cloning inserts, and characterize unknown DNA.
What is RFLP analysis?
Restriction Fragment Length Polymorphism (RFLP) analysis uses differences in fragment sizes between individuals (due to SNPs that create or destroy recognition sites) for genetic fingerprinting, paternity testing, and disease gene mapping. Different individuals may have different restriction patterns at the same locus.
What units should I use for cut positions?
Enter cut positions and total DNA length in the same units: typically base pairs (bp) for small DNA, or kilobases (kb) for larger sequences. The calculator simply subtracts positions, so units do not matter as long as they are consistent throughout.
Official sources
- NCBI: NCBI restriction enzyme and primer tools.
- NIH/NHGRI: Restriction Enzyme definition.
Reviewed by the CalculatorHub team, edited by James Graham, 15 June 2026. See our methodology.