Gel electrophoresis

Why it matters

Agarose gel electrophoresis is the quickest way to see DNA. After a PCR, a restriction digest or an extraction, a gel tells you whether you have a product, how big it is, whether there is one band or several, and whether your controls behaved. Reading a gel correctly, and photographing and recording it properly, is a daily skill for biological technicians and an early step in many clinical molecular tests.

The core idea

DNA has a negatively charged phosphate backbone, so in an electric field it moves toward the positive electrode (anode). A common lab saying is "run to red", because the positive lead is usually red. An agarose gel is a mesh: small fragments slip through quickly, large fragments are slowed. After a run, fragments are sorted by size, smallest farthest from the wells.

To see the DNA, the gel contains (or is soaked in) a fluorescent stain. Many teaching labs use safer stains viewed with blue light; some labs still use ethidium bromide with UV light, which needs specific training and protection because the stain is a mutagen and UV burns skin and eyes. Either way, gel work is done in a lab with your instructor or supervisor, never at home.

Choosing a gel

Higher percentages resolve small fragments better. The gel and the tank use the same running buffer (TAE or TBE).

The ladder

A DNA ladder (size marker) is a mix of fragments of known sizes, loaded in its own lane. It is your ruler. Over the useful middle of the gel, the distance a fragment travels is roughly linear in the logarithm of its size. So to size an unknown band:

1. Measure the migration distance of each ladder band from the bottom of the well.
2. Plot log10(size in bp) against distance and draw the best straight line through the bands near your unknown.
3. Read the unknown's size from its distance.

Worked example. Ladder bands at 1,000 bp (18 mm), 750 bp (22 mm), 500 bp (28 mm) and 250 bp (37 mm). Your PCR band is at 25 mm, between the 750 and 500 bp bands. Interpolating in log space: log10(750) = 2.875 at 22 mm and log10(500) = 2.699 at 28 mm. At 25 mm, halfway, log10(size) is about 2.787, so the size is about 610 bp. If your predicted product is 600 bp, that is a match. (Simply eyeballing "between 500 and 750" is often enough for a yes/no PCR check.)

Reading a whole gel

Go lane by lane and write down what you see before you interpret it:

• Expected lanes: one sharp band at the predicted size.
• No-template control: empty. A band here means contamination.
• Positive control: the expected band. If it is missing, the run failed, not your samples.
• Extra bands: non-specific products or primer dimers (a faint, fuzzy band below about 100 bp).
• Smears: degraded DNA, too much DNA, or too many PCR cycles.
• Uncut plasmid can show several bands (supercoiled, nicked, linear) that run at different apparent sizes from the same molecule, which is why you size linear fragments against a linear ladder.

Recording the result

Save the original gel image without editing it (adjusting overall brightness is usually allowed, editing individual bands never is). Label lanes, the ladder and its sizes, the gel percentage, the date and your initials, and attach the image to your notebook entry. This is the same data integrity you learned for any lab record.

Common mistakes

• Running the gel backwards, so the DNA runs off the top into the buffer.
• Sizing against the wrong ladder (check which ladder lot you loaded and its band sizes).
• Reading sizes from the far ends of the gel, where the log-linear relationship breaks down.
• Skipping the controls when interpreting the samples.
• Cropping or editing images in ways that hide bands.

Quick recap

• DNA runs toward the positive electrode; small fragments go farthest.
• Choose agarose percentage by fragment size.
• Size bands by interpolating log10(bp) against distance from a ladder.
• Check NTC and positive control first; record unedited, labelled images.

Try it

Open the Do tab to size bands from a set of gel measurements on paper or graph paper.