Agarose gel electrophoresis for DNA

Agarose gel electrophoresis answers a size question. DNA is pulled through a polysaccharide mesh by an electric field, and shorter fragments thread the mesh faster than longer ones. A molecular weight ladder of known lengths turns the distance travelled into an estimate. The estimate is often good enough to accept a PCR product, to check a restriction digest, or to decide which slice of a gel to cut out. It is not a sequence, and it is not a protein blot. Protein transfer is a different method, described in western blot from gel to membrane.

This page is a research explainer for reading and planning that DNA gel. It is not a clinical test and not a stain manufacturer's insert. Matrices, markers and tanks are sourced from the molecular biology catalogue, with the scientific requirement attached to the quote request.

What the gel is sorting

In a typical horizontal gel the sample wells sit toward the negative electrode, the cathode. DNA carries a negative charge from its phosphate backbone, so it moves toward the anode. Agarose concentration sets the mesh. Laboratories often start near 0.7 to 0.8 percent when the fragments are many kilobases, near 1 percent for ordinary plasmid and PCR work, and near 1.5 to 2 percent when a small amplicon must be separated from primer-dimer. Those are starting windows, not a table copied from one vendor. A fragment that is wrong for the percentage stacks with its neighbours and the ladder cannot save the interpretation.

The running buffer carries the current and keeps the pH from drifting. Tris-acetate-EDTA and tris-borate-EDTA behave differently as they heat, and borate can matter if DNA is later removed from the gel for cloning. Use one family for the gel and the tank. A tired tank buffer slows the run and makes ladders look unfamiliar. Replace it rather than guessing at a higher voltage.

The ladder is a set of linear fragments of stated sizes. It belongs on the same gel, in the same buffer, photographed in the same exposure. A pretty ladder from another day is not a scale bar. Choose a ladder whose bands flank the size you care about. A ladder that stops at 1 kilobase cannot measure a 3 kilobase band except by waving a hand.

Loading, voltage and the photograph

Molten agarose is a burn hazard. Cast it after it has cooled enough to handle, with a comb that does not touch the tray floor. A torn well leaks into the next lane. Loading dye adds density and visible markers of progress. Those dyes are not size standards. Stop the run because the separation you need has happened.

A field that is too high warms the centre of the gel and the lanes curve. In a hot room the same voltage can soften the agarose until bands blur. Lower the field and give the run more time. A power cut mid-run lets bands diffuse. If they have sat, repeat the gel rather than trusting the resumed distances.

Stain binds nucleic acid so you can see it. Ethidium bromide and newer alternative dyes are different hazards and different sensitivities. Follow the safety note for the bottle you actually have. View the gel through the shield on the imager. Ultraviolet light damages eyes, skin and the DNA you may want to clone. Photograph, then cut the band you intend to ligate.

What a lane can honestly say

A single sharp band near the expected size, with a clean no-template lane on the same gel, supports "a product of about this length was made". How those PCR controls are read is covered in PCR controls and contamination control. A second band means a second species, a partial digest, a primer-dimer, or an overloaded well that smeared. Primer-dimer sits low on the gel and should not be cut out by accident with a small amplicon. Give the run enough distance that those two are not one glow.

A smear from the well down the lane can be degraded DNA, too much DNA, or salt and protein carried from a crude prep. Dilute the load before you conclude the sample is ruined. DNA stuck in the well often means it is very long, or it is bound to something, or the well itself is blocked. Genomic DNA that was meant to stay long should not be judged by the rules you use for a 400 base-pair PCR band.

Uncut plasmid is the classic mis-read. Supercoiled circles slip through the gel faster than a linear fragment of the same sequence. Relaxed circles lag. If you need the length, cut with an enzyme that linearises once, and then compare with the ladder. A restriction map is only as good as a complete digest. A partial digest invents extra bands and looks like the wrong clone.

Quantity is a different experiment. Band brightness saturated by the stain or by the camera is not a standard curve. If you need mass, load a mass ladder or use a method designed for quantification. If you need purity of a single species, a gel can show that a second species of different size is absent. It cannot show that two sequences of the same size are absent.

Failure modes

No bands at all, including the ladder, means the field was reversed, the stain failed, the photograph was taken on the wrong setting, or the gel was never actually run. Check the electrodes and the stain before you throw away the samples. No sample bands with a healthy ladder means the DNA never entered the well, the load was too small, or you ran a small product off the end. The tracking dye is the clue you should have used earlier.

Fuzzy ladders after a buffer change usually mean the percentage, the buffer family, or the temperature changed. Pour a fresh gel before you redesign the biology.

Safety and the room

Ultraviolet transilluminators and ethidium bromide, or whichever stain you use, are the routine hazards. The orange shield in front of a gel is there to be used, not to be tilted aside for a better look. Gloves come off before you touch doors and notebooks. Disposal of stained gels follows the rule your institution set, which may be stricter than a product page. The WHO laboratory biosafety manual is a public reference for how laboratories frame chemical and biological risk. It does not replace your local rule.

Heat is the local variable that changes electrophoresis. Buffer that starts warm, a closed tank in a hot afternoon, and a high voltage combine to melt resolution. If runs suddenly smile in one season, lower the field before you blame the polymerase. After a power cut, look at the gel before you trust the distances. Diffusion is invisible until the bands are compared with a ladder that also sat there.

What to ask for

EVRINTH can take a sourcing question about agarose, markers, stains and tanks as catalogue classes. State the fragment sizes you must resolve, whether DNA will be recovered from the slice, and the buffer family you have standardised on. The nucleic acid analysis pathway is the context when the gel sits between a PCR and a clone or a sequencing reaction. Ask whether a quotation is possible. Do not treat a ladder range printed in a title as a promise that every fragment between those numbers is resolved on your percentage.