How to Predict an Agarose Gel From a Restriction Digest

6 min read · Updated July 10, 2026

Running a restriction digest and getting a band pattern you cannot explain wastes a gel and a day. Before you cut, you can calculate exactly how many fragments a given enzyme, or combination of enzymes, will produce and how large each one will be, then work out what the resulting agarose gel should look like next to a DNA ladder. This guide covers the math behind fragment prediction for linear and circular templates, why gel migration is not linear with fragment size, and how to pick an agarose percentage that will actually resolve the bands you expect.

Start with cut positions, not just an enzyme name

An enzyme name tells you a recognition sequence, but predicting a digest requires the actual cut positions along your specific template. For each enzyme in the digest, find every site that matches its recognition sequence and note the coordinate where it cuts. You also need to know whether the template is linear (a PCR product, a linearized fragment) or circular (an intact plasmid), because that changes how cut positions turn into fragment counts and sizes.

Counting fragments: linear templates vs circular plasmids

Once you have every cut position for the enzyme or enzymes in the digest, the fragment sizes follow directly from simple arithmetic, but the rule differs by topology.

Linear template: sort all cut positions in ascending order along the sequence.
Linear template: each internal fragment is the distance between two consecutive cuts.
Linear template: add two end fragments — from the start of the sequence to the first cut, and from the last cut to the end of the sequence.
Circular plasmid: the number of fragments equals the number of cuts, since there are no free ends.
Circular plasmid: fragment sizes are the distances between consecutive cuts going around the circle, including the distance from the last cut back to the first, wrapping across the origin.
Circular plasmid: as a check, all fragment sizes must sum to the total plasmid length.

Why fragment size does not translate directly into gel distance

Migration through agarose is roughly inversely related to the log of fragment size: larger fragments migrate slower and travel a shorter distance from the well, while smaller fragments migrate faster and travel farther, so a 200 bp difference matters a lot at the low end of a gel and barely at all between two large fragments. This is why a 1 kb and a 10 kb fragment are much easier to tell apart than a 5 kb and a 6 kb fragment on the same gel, and why band spacing on a real or predicted gel image is usually reasoned about on a log scale rather than a linear one.

This is also why a DNA ladder, a marker lane with bands of known size, is run alongside every gel: it is the actual calibration for reading distance back into size, since migration distance alone is not a reliable size scale without it.

Uncut plasmid is not a size reference

An intact circular plasmid, especially in its supercoiled state, does not migrate at a position consistent with its actual base-pair length. Supercoiled DNA is more compact and runs faster than the same molecule linearized, so it looks smaller than it is, while relaxed or nicked circular DNA runs slower and looks larger.

A predicted gel built from linear fragment sizes is therefore only directly comparable to a real gel for the linearized fragments produced by a digest, not for an uncut plasmid lane, which will not land where its length would suggest.

Picking the right agarose percentage before you run anything

Agarose percentage sets the size range a gel resolves well, and predicting fragment sizes ahead of time tells you which percentage is worth pouring. Lower-percentage gels, roughly 0.7 to 1 percent, resolve larger multi-kilobase fragments better, while higher-percentage gels, roughly 2 to 3 percent, resolve small fragments under about 500 bp that would otherwise run together near the bottom of a standard gel.

If your predicted digest produces a mix of very large and very small fragments, that is a sign one gel percentage will not show all of them clearly, and you may need to decide which fragments matter most or run two gels.

Predicting the gel before you run one

Working through cut positions, fragment arithmetic, and log-scale migration by hand is tedious to redo for every construct and every enzyme combination. Virtual Gel takes a sequence and a chosen enzyme or combination of enzymes, computes the resulting fragments for a linear or circular template, and shows the simulated agarose gel with a ladder so you can see the expected band pattern before touching a pipette.

That predicted pattern is a reference: it confirms the construct you have matches the digest you expect by band count and size, flags in advance when two fragments will be too close in size to resolve on a normal gel, and gives you something concrete to compare against if a real digest turns up extra or missing bands, which usually points to partial digestion or star activity.

To find the cut sites feeding that prediction, Restriction Sites lists recognition and cut positions for common enzymes on your sequence, and Plasmid Viewer renders those same sites on a circular or linear map so you can see where each cut falls relative to your construct's features.

Frequently asked questions

How do I calculate restriction fragment sizes without running a gel?

List every cut position for the enzyme or enzymes in the digest, then for a linear template take the distances between consecutive cuts plus the two end fragments. For a circular plasmid, take the distances between consecutive cuts all the way around, including from the last cut back to the first.

Why does my uncut plasmid run at a different size than expected on a gel?

An intact plasmid is usually supercoiled, which runs faster and looks smaller than its actual length, while a nicked or relaxed circular form runs slower and looks larger. Only linearized digest fragments migrate at a size consistent with their base pair length.

What agarose gel percentage should I use for small DNA fragments?

Fragments under about 500 bp resolve best on higher-percentage gels, roughly 2 to 3 percent agarose, while multi-kilobase fragments resolve better on lower-percentage gels around 0.7 to 1 percent.

How many fragments does a restriction digest of a circular plasmid produce?

Exactly as many fragments as there are cut sites, since a circular molecule has no free ends. A plasmid with three cut sites for your enzyme combination will always produce three fragments whose sizes sum to the total plasmid length.