DNA ladder selection: range, reference bands and loading

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.

Match the ladder's range to the question

A ladder is useful only where its bands bracket the fragment of interest, and its resolution is best in the middle of its range. A wide-range set is convenient and resolves poorly at both ends, so a laboratory running one size class routinely is better served by a ladder centred on it.

The gel matters as much: a percentage that resolves small fragments compresses large ones into a single band, so the ladder and the gel are chosen together. Reporting a size from a compressed region is an estimate with a wide error.

Denaturing gels need a single stranded DNA ladder

On a denaturing gel a double-stranded marker runs as separated strands of a different apparent size, so it cannot be used to size a single-stranded product. A ladder supplied as single strands of known length is what the gel requires.

This matters for oligonucleotides, for RNA and for anything where secondary structure would otherwise change mobility. Using the wrong marker gives sizes that are confidently and consistently wrong.

Lambda DNA: a standard genome as a reagent

A bacteriophage genome of known sequence and length is used as a quantification standard, as a substrate for testing enzymes and nucleases, as a carrier and as a control template. Its value is that its length and sequence are exactly known.

It is also the source of several traditional ladders, which is why digests of it appear as markers. For quantification against a fluorescent dye it remains a convenient, well-characterised standard.

A dna page gel, and when polyacrylamide beats agarose

Agarose resolves fragments from about a hundred bases upward and is poured in minutes, which is why it carries almost all routine work. Polyacrylamide resolves single base differences under a few hundred bases, which is what oligonucleotide purity, footprinting, small RNA and heteroduplex work need, and it runs in a vertical cast gel with the handling that implies. The ladder has to match: a low range set with closely spaced bands, run and stained the same way as the sample, since a prestained agarose ladder means nothing on an acrylamide gel.

A 10 kb dna ladder and reading large fragments

A 10 kb dna ladder resolves the range where agarose separation is losing power, so the gel percentage, the run time and the voltage decide whether the top bands separate at all, and a lower percentage gel run slowly is what makes them readable. For anything larger the answer is pulsed field or capillary sizing rather than a longer run.

tricine gels and the peptides they resolve

tricine gels replace glycine in the running buffer so that small peptides and proteins resolve instead of running off the bottom, which is why they appear in membrane protein and peptide work. The trade is a longer run and a different transfer, since small peptides pass through a standard membrane, and fixing before staining is what keeps them in the gel.

An sds-page power supply and what it has to hold

An sds-page power supply is specified by whether it can hold constant voltage, current or power and by its maximum in each, because the mode chosen changes the run: constant voltage is usual for a gel, constant current for a transfer. Timer and automatic shutoff matter for an overnight run, and a supply without a safety interlock has no place beside a full tank.

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.

Which ladder range should I buy?

One whose bands bracket your fragment, since resolution is best mid-range. A wide-range set is convenient and resolves poorly at both ends; choose the ladder and the gel percentage together.

Can I use a double-stranded ladder on a denaturing gel?

No. Its strands separate and run at a different apparent size, so sizes read from it are wrong. Use a ladder supplied as single strands of known length.

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