Reading ladders bands and smears

A ladder is the only size scale that belongs to a gel. The bands and smears in the sample lanes mean something only while that scale is on the same photograph, run in the same buffer, on the same percentage. This page explains how to read that picture: why mobility is roughly logarithmic, why a mark outside the ladder has no honest length, and how thick bands, doublets, an RNA smear and primer-dimer each ask a different question. How the gel is poured and run is agarose gel electrophoresis for DNA. This is a research explainer. A lane is not a sequence and not a diagnosis.

Markers, trays and stains are catalogue classes in the molecular biology catalogue. The sizes you must bracket belong on the quote request.

What you are allowed to claim from a lane

You are allowed to compare a sample feature with ladder rungs that flank it on that image. You are allowed to say a lane is empty, smeared, overloaded, or holding more than one species. You are not given a length for a band that sat above the slowest rung or below the fastest rung. You are not given identity. Two sequences of the same length are one band. Write the claim in those limits before you crop the photo for a notebook.

The expected length comes from the map or the reference sequence you actually amplified or cut. NCBI Nucleotide is a public place to check a recorded sequence length. Your construct wins if you added or deleted bases the record does not have. The ladder then tests whether the dominant species migrated like that plan.

Why the spacing is logarithmic

DNA in a typical agarose gel carries a roughly constant charge for its length, so the field pulls fragments in proportion to size while the mesh drags longer ones more. Across the middle of a well-chosen percentage, the distance travelled falls about linearly as the logarithm of length increases. Equal steps on the ladder card are therefore unequal steps on the gel. The big fragments bunch nearer the wells. The small ones spread, until the percentage is so loose that they pile up again at the front.

That shape has two practical consequences. Between two flanking rungs, a sample band can be interpolated, and a log mental model is fairer than a ruler that pretends the gel is linear. Outside those rungs, interpolation becomes invention. The curve flattens near the wells and compresses again near the dye front. A band "a bit above" the top rung is larger than that rung. It is not 12 kilobases because the top rung was 10 and the gap looked like a fifth of the way to an imaginary 20.

Do not repair a missing rung by borrowing one from another gel. Percentage, buffer, temperature, voltage and time all move the curve. A ladder photographed last week is a picture of a different curve.

Thick bands, doublets and shapes that are not linear

A thick band is a distribution, not a line. The brightest core is the usual place to compare with the ladder. The leading and trailing fuzz are not extra sizes unless a lighter load splits them into two cores. Doublets are already split. Averaging the pair into one "about" size hides the fact you have two answers. Partial restriction digests invent those extra bands. So do two PCR products the primers were capable of making.

Uncut plasmid breaks the log story on purpose. Supercoiled, relaxed and linear forms of one sequence do not travel as one linear length. The pillar page is the place for that reading. If the question is length, linearise, then use the ladder. If the question is "did I recover circular plasmid," the extra forms are the observation, and you still do not quote them as base pairs from a linear card.

An RNA smear in a genomic preparation

Genomic DNA that survived extraction should be high in the lane, close to the well, as a slow band rather than a cloud from top to bottom. A smear can mean the DNA was sheared. It can also mean RNA came through the prep and stained. Ribosomal RNA is abundant. On a native agarose gel it often appears as a broad smear or as bands in the middle of the lane, and it is easy to call that "degraded genome" because both pictures are messy.

Split the question. Load less. If the high band is still there and the smear thins, overload was part of the story. Treat a matched aliquot with an RNase of the class your laboratory already uses, following that enzyme's card and the chemical rules around it, and run it beside the untreated lane. If the smear goes and the high DNA remains, the smear was RNA. If both the high band and the smear collapse, you are looking at nuclease damage or a reaction that was not specific, and the tube is not a clean genome prep. This article does not give a volume recipe for that test.

Loading dye still only marks progress. It will not tell RNA from DNA. A transfer membrane is irrelevant to this reading. The smear is a gel observation. Blotting it would copy the ambiguity onto another sheet.

Primer-dimer at the dye front

Loading dye adds density so the sample sinks, and it adds coloured markers that move ahead of most of the DNA you care about. Bromophenol blue and xylene cyanol are the usual pair. Where they sit, in base pairs, depends on the agarose percentage. People who memorise "the blue dye is 300 base pairs" on one gel mis-stop the next percentage.

Primer-dimer is short double-stranded DNA made from the primers alone. It travels with or just behind the fastest dye, and it is often fuzzier than a designed amplicon. On a loose gel it merges with a small product into one glow at the front. The no-template lane is what convicts it. If that lane glows at the same place, the sample lane cannot be called a clean product, even when a brighter band sits higher up. The controls note is PCR controls and contamination control. Cut a slice only from a band you can point at between ladder rungs, not from the dye.

A photograph without the ladder is an incomplete record

The image is the data. If the ladder lane is cropped out, blown to a white block, hidden by a reflection, or never loaded, the file cannot be sized later by memory. Write the ladder's identity and the agarose percentage on the record next to the file. Keep a frame in which the rungs you will cite are visible as separate marks, not a single saturated stripe. The orange shield in the photograph is there so you can make that frame without looking at a bare ultraviolet box. Ultraviolet light damages eyes, skin and the DNA in the gel.

A smear that fills the lane from well to dye is also a record. Do not retake the picture at a shorter exposure until the smear "goes away" and then file only the pretty frame. The short exposure may hide the evidence you needed. Save a frame in which both the ladder and the shape of the smear are still visible.

Safety, and what the reading is not

Stains and ultraviolet light are the hazards of looking. Follow the safety data for the stain bottle in your hand. The electrical run is already finished by the time you read. Biosafety is the sample's containment, decided by the institution. Nothing in a band pattern authorises a diagnostic or forensic claim.

A clean interpolation does not survive a gel that melted, a field that was reversed, or a buffer you changed without saying so. If the ladder itself is curved or fuzzy, every sample lane inherits that doubt. Read the ladder first. If you would not trust its rungs, do not quote a sample size beside them.

Humidity on the shield, and a run that sat

In a humid room the orange shield and the camera window fog. Faint ladder rungs disappear first, and the file looks like a gel with no scale. Wipe the shield, confirm the rungs are back, and expose again before you call a lane negative or smeared. A power cut that leaves the gel sitting lets bands and rungs diffuse together. That photograph can show that something fluorescent was there. It cannot support a length. Repeat the run rather than drawing log ticks on a softened ladder.

What to put in an enquiry

Name the size window you must bracket, the percentage you run, and whether the awkward feature is a close doublet, a small amplicon against primer-dimer, or a genomic prep you suspect contains RNA. Say if you need a mass ladder as well as a size ladder. Those are different cards. The nucleic acid analysis pathway is the context when the gel sits between a prep and a clone or a sequencing check. Ask whether a quotation is possible for the marker range and the stain class you named. A ladder title that prints a wide range is not a promise that every interval inside it is resolved on your mesh.