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.

Plasmid Insert Size Estimator

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 N fragments. A single cut produces one full-length linear band, and no cuts leave an uncut circle. Fragments are reported largest first to match how they migrate on a gel.

Worked example

A 3000 bp vector with a 1500 bp insert is a 4500 bp circle. Cutting at positions 500, 2200, and 4000 gives gaps of 1700 bp (500 to 2200) and 1800 bp (2200 to 4000), plus a wrap-around fragment of 4500 minus 4000 plus 500, which is 1000 bp. The expected bands are 1800, 1700, and 1000 bp.

Tips

Choose enzymes that cut asymmetrically so a correct clone gives a clearly different pattern from empty vector. Watch for fragments under about 100 bp, which can run off the gel or fade, and for similarly sized bands that may not resolve. Always run a marker ladder alongside to read sizes accurately.