Why does a plasmid give one fewer band than I expect?

A plasmid is circular, so N cut sites produce exactly N fragments, not N plus one. The fragments are the gaps between consecutive sites, and the final fragment wraps around the origin from the last site back to the first, which the calculator handles automatically.

What happens with a single cut site?

One cut simply opens the circle into a single linear molecule, so you see one band at the full plasmid size. This is how you linearise a vector. With no sites at all the plasmid stays an uncut supercoiled circle.

How do I find the cut positions?

Map your plasmid sequence against the recognition sites of your chosen enzymes, either by eye or with a restriction mapping tool, and read off the base pair coordinate of each cut. Enter those coordinates here as positions around the circle.

Why use a diagnostic digest at all?

After ligation and transformation you need to confirm the insert is present and in the right orientation. A digest that cuts asymmetrically across the insert and backbone produces a characteristic band pattern that distinguishes a correct clone from empty vector or wrong orientation.

Does fragment order on the gel match the list?

Larger fragments migrate more slowly and sit higher on the gel, while small fragments run to the bottom. The tool sorts bands largest first to mirror that top-to-bottom order, though very small fragments may run off the gel or be hard to see.

What is the Plasmid Insert Size Estimator?

Takes vector and insert sizes plus restriction cut positions on a circular plasmid and predicts the diagnostic digest fragment sizes, accounting for the circular wrap-around fragment. For confirming clones in molecular cloning. It runs free in your browser on Gera Tools, with nothing uploaded.

Plasmid Insert Size Estimator

Name: Plasmid Insert Size Estimator
Creator: Gera Tools
License: https://creativecommons.org/licenses/by/4.0/

Get one useful tool a week

Like this tool? Enter your email and we'll send you one genuinely useful Gera tool a week — plus a link to come back to this one. No spam, one-click unsubscribe any time.

Confirming a clone usually means running a diagnostic restriction digest and checking the band pattern. This estimator predicts exactly which fragment sizes to expect from a recombinant plasmid, correctly treating the molecule as circular so the fragment count is right.

How it works

The total recombinant plasmid size is the vector backbone plus the insert. Cut positions are coordinates around that circle. The tool sorts the positions and takes the gap between each consecutive pair, then adds the wrap-around fragment running from the last site, through the origin, back to the first site.

Because the molecule is circular, N cut sites always yield exactly N fragments. A single cut produces one full-length linear band, and no cuts leave an uncut supercoiled circle that runs aberrantly on a gel. Fragments are reported largest first to match how they migrate from top to bottom on an agarose gel.

Worked example

A 3,000 bp backbone with a 1,500 bp insert gives a 4,500 bp recombinant circle. Choosing EcoRI (cuts at position 500) and HindIII (cuts at positions 2,200 and 4,000 — for example) gives three sites:

Site	Position
Cut 1	500 bp
Cut 2	2,200 bp
Cut 3	4,000 bp

Gaps: 2,200 − 500 = 1,700 bp, 4,000 − 2,200 = 1,800 bp, and the wrap-around = 4,500 − 4,000 + 500 = 1,000 bp. Expected bands: 1800, 1700, 1000 bp.

A correct clone gives this pattern; empty vector (3,000 bp only) would give bands at 2,500 and 500 bp — a clearly distinguishable result that confirms the insert is present.

Designing a good diagnostic digest

Cut asymmetrically across the insert. Choosing sites that bracket the insert from different distances distinguishes correct orientation from reverse-orientation inserts, which give a swapped band pattern.
Aim for bands that resolve well. Fragments closer than about 100 bp in size are difficult to distinguish on a standard 1 % agarose gel. If two expected bands are very close, choose a different enzyme pair.
Watch for very small fragments. Fragments under roughly 100 bp run off the gel quickly and may be invisible. Include them in your mental map but do not rely on them for confirmation.
Always run a ladder. A 1 kb or 100 bp DNA ladder alongside lets you size bands accurately. Band size estimation by eye without a marker is unreliable.
Uncut control. Running an uncut sample beside the digest lets you confirm your supercoiled, nicked circular, and linear forms are where expected, which helps diagnose nicking or degradation.
Sequence-verify before relying on the digest. A diagnostic digest confirms the insert is present and roughly the right size, but it does not verify the sequence. For critical constructs always follow up the colony screen with Sanger sequencing to confirm there are no PCR-introduced mutations in the insert or at the ligation junctions.