Restriction Enzyme Digest Calculator

How this restriction enzyme digest calculator works

This tool performs a virtual restriction digest on your DNA sequence. It scans for recognition sites of one or two restriction enzymes, computes the cut positions, and then derives the resulting fragment sizes and a simple restriction map.

1. Cleaning and parsing your DNA sequence

The calculator accepts plain DNA sequences or FASTA-formatted input. Any header lines starting with > are ignored, and only the characters A, T, G, and C (case-insensitive) are kept. All other symbols (spaces, numbers, ambiguous bases) are removed before analysis.

2. Recognition sites and cut positions

Each restriction enzyme is defined by:

• Name (e.g., EcoRI, HindIII)
• Recognition sequence (e.g., GAATTC)
• Cut offset: the 0-based index of the phosphodiester bond cut relative to the 5′ end of the recognition site on the top strand.

For simplicity, this calculator assumes a single cut per recognition site and does not model staggered vs. blunt ends in detail; it focuses on fragment boundaries and sizes.

3. Fragment size calculation

After finding all cut positions for the selected enzymes, the tool sorts them and computes fragment lengths.

Linear DNA

For a linear molecule of length \(L\) with cut positions \(c_1, c_2, \dots, c_n\) (0-based, sorted), the fragment lengths are:

• First fragment: \( f_1 = c_1 - 0 \)
• Intermediate fragments: \( f_i = c_i - c_{i-1} \) for \( i = 2, \dots, n \)
• Last fragment: \( f_{n+1} = L - c_n \)

Circular DNA

For a circular plasmid, the ends are joined, so the last fragment wraps around:

• Fragments: \( f_i = c_i - c_{i-1} \) for \( i = 2, \dots, n \)
• Wrap-around fragment: \( f_{\text{wrap}} = L - c_n + c_1 \)

4. Single vs. double digest

In a single digest, only one enzyme is used; all cut positions come from that enzyme. In a double digest, the calculator:

1. Finds all recognition sites for enzyme 1 and enzyme 2.
2. Combines and sorts all unique cut positions.
3. Applies the fragment formulas above to the merged list.

This mirrors how a real double digest generates a fragment pattern determined by the union of both enzymes’ cut sites.

Practical tips for planning a restriction digest

While this calculator focuses on the in silico map, successful wet-lab restriction digests also depend on buffer conditions, enzyme activity, and DNA quality.

• Check buffer compatibility: For double digests, choose a buffer where both enzymes have high activity (consult the manufacturer’s chart).
• Use enough units of enzyme: A common rule of thumb is 1 unit per µg DNA for 1 hour in optimal conditions.
• Avoid star activity: Excess enzyme, long incubations, or non‑optimal buffers can cause off‑target cuts.
• Consider methylation sensitivity: Some enzymes do not cut methylated sites (e.g., dam/dcm methylation in E. coli).

Limitations of this restriction digest calculator

• Assumes exact recognition sequences (no degenerate bases like N, R, Y).
• Does not model methylation, star activity, or partial digests.
• Does not distinguish between 5′ overhang, 3′ overhang, or blunt ends beyond reporting fragment boundaries.

For critical cloning steps, always cross‑check with the enzyme supplier’s documentation and, if possible, validate your construct by sequencing.