BioDeviceHub

Restriction Digest Predictor

Predict the fragment sizes of a restriction digest with one to four enzymes on a linear or circular DNA, for planning a diagnostic digest or a double digest.

Formula

linear: n cuts give n+1 fragments\text{linear: } n \text{ cuts give } n + 1 \text{ fragments}
circular: n cuts give n fragments (one cut linearises the plasmid)\text{circular: } n \text{ cuts give } n \text{ fragments (one cut linearises the plasmid)}
fragment size=difference between successive cut positions\text{fragment size} = \text{difference between successive cut positions}
cut position\text{cut position}
bases of the top strand to the left of a cut
size\text{size}
length in base pairs of the top strand between two cuts

How it works

Each enzyme's sites are found, every cut position is collected, and the lengths between successive cuts are the fragments. For a circular molecule the last fragment wraps around the origin, so cuts equal fragments. Using two enzymes together (a double digest) simply pools their cut positions.

The fragments are listed largest first, which is the order they appear on an agarose gel from the top. Fragments of the same size appear as a single band, which the tool points out, because it can hide a fragment you expected to see.

Worked example

A 115 bp linear sequence containing two EcoRI sites and two HindIII sites, cut with both enzymes.

  1. Four cuts give five fragments.
  2. The largest lies between a HindIII and an EcoRI cut, 51 bp.

Fragments of 51, 19, 18, 14 and 13 bp, which add up to 115.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • Complete digestion, with every site cut. Partial digests give extra bands.
  • The enzymes are active in a common buffer; some pairs need sequential digestion.
  • Sizes are for the top strand; staggered ends add a few bases of overhang to each fragment.

Common mistakes

  • Expecting a band for a tiny fragment, which can run off the gel or stain too faintly to see.
  • Forgetting that supercoiled, nicked and linear forms of an undigested plasmid run differently, so uncut DNA shows several bands.
  • Reading the sizes without checking whether the plasmid is linear or circular.

Related tools

Restriction Site FinderSites, cut positions and overhangs for named restriction enzymesOpen calculatorRestriction Digest Reaction SetupVolumes of DNA, buffer, enzymes and water for a digestOpen calculatorLigation Calculator (Vector:Insert Ratio)Insert mass for a molar ratio, or the ratio from the masses you haveOpen calculatorPlasmid Size CalculatorSize of a recombinant plasmid after cloningOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.