dna ladder selection: matching the size range to the gel, using a reference band for quantitation, and why ladders stop working

Choosing and using it

1. Match the range to the fragments. A ladder spanning a wide range has coarse spacing in the middle; one designed for small fragments resolves them finely and says nothing above its top band. Choose from the sizes you actually assign, and keep a second ladder for the other end rather than extrapolating.
2. Spacing matters more than the number of bands. Closely spaced bands around your fragment size let you assign a length with confidence; a ladder whose nearest bands are far apart gives you a range, not a size. Look at the spacing near your region of interest rather than the band count.
3. Reference bands give you quantity. Many ladders load a known mass into designated bands, so comparing your band's intensity to a reference band estimates quantity as well as size. It is approximate and it is free, and it is the quickest sanity check on a preparation's concentration.
4. Ready-to-load or concentrate. Ready-to-load formats include loading dye and are quicker and more consistent; concentrated stocks are cheaper per lane and need dilution and dye. For routine work the ready format usually pays for itself in avoided pipetting errors.
5. Store it properly and replace it. Repeated freeze and thaw cycles and prolonged warmth degrade a ladder, and a smeared or partial ladder makes every gel it appears on uninterpretable. Aliquot on arrival, and discard a ladder that no longer runs cleanly rather than working around it.

Reading a gel honestly

Migration is affected by gel percentage, buffer, voltage, running time and the conformation of the DNA, so a supercoiled plasmid does not run at the size a ladder would suggest. Size assignment from a gel is an estimate, and where the exact length matters the answer comes from sequencing.

Run the ladder on the same gel under the same conditions, never from a previous gel, and photograph both together. A ladder from another run is decoration.

Ladders for other separations

Capillary and automated electrophoresis systems use their own internal standards rather than a lane of ladder, and the sizing is generally more accurate. Where exact sizing matters routinely, that is usually a better instrument choice than a better ladder.

For RNA, a dedicated RNA ladder run under denaturing conditions is required; a DNA ladder run on an RNA gel gives misleading positions.

Common questions

How do I choose a DNA ladder?

Match its range and, more importantly, its band spacing to the sizes you actually assign. A wide-range ladder has coarse spacing in the middle, which turns a size assignment into a guess.

Can I use a DNA ladder to estimate concentration?

Approximately, where the ladder loads a known mass into designated bands. Comparing your band's intensity to a reference band is a quick and free sanity check, though not a substitute for proper quantitation.

Why does my plasmid not run at its expected size?

Conformation changes migration, so supercoiled, relaxed and linear forms of the same plasmid run differently. Linearise before sizing, and treat gel sizing as an estimate.

Why has my ladder stopped giving clean bands?

Usually degradation from repeated freeze and thaw cycles or prolonged warmth. Aliquot on arrival and discard a ladder that no longer runs cleanly rather than interpreting around it.

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