Gel Electrophoresis

Why size and not charge

Every phosphate carries a negative charge, so DNA moves toward the positive electrode (anode), and its charge grows in step with its length ¹. That makes charge-per-length roughly constant, and separation comes from the gel: a mesh that lets small fragments slip through faster than large ones ². Load at the negative end (wells near the black cathode) - “run to red.”

Agarose or polyacrylamide

Agarose percentage tunes the range ³: about 0.7% for large fragments (several kb), 1% for 0.5-10 kb, 2% for 100-1,000 bp, and 3% for small PCR products. Higher percentage, smaller pores, better separation of small pieces.

Buffers

TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) carry current and hold the pH so the backbone stays charged. TAE separates large fragments a bit better; TBE buffers longer and resolves small fragments more sharply ³. EDTA chelates the Mg²⁺ nucleases need. Use the same buffer for the gel and the tank.

Loading dye adds density (glycerol) so the sample sinks into the well, plus tracking dyes to watch progress.

Sizing with a ladder

Over a gel’s working range, migration distance is roughly linear with the log of size ³. A ladder of known fragment sizes in its own lane lets you interpolate: find the two ladder bands that bracket the sample and estimate between them, remembering that on a log scale the bands crowd together toward the top ².

Band brightness reflects mass: a ladder with stated masses per band lets you roughly estimate how much product you have.

Stains

Fluorescent dyes bind between or alongside the bases and glow under UV or blue light ³:

• Ethidium bromide: classic, cheap, viewed under UV; a mutagen, so gloves and dedicated waste.
• SYBR and GelRed-type dyes: more sensitive, many viewable under blue light, marketed as safer.
• UV safety: face shields for transilluminators; UV also nicks DNA you plan to recover from the gel, so minimize exposure (blue light avoids this).

Reading a gel

²

Uses

• Checking PCR products before sequencing or cloning.
• Restriction digests and PCR-RFLP genotyping (see Restriction Enzymes and RFLP).
• Integrity of DNA and RNA (see Assessing Quality and Quantity).
• Gel extraction: cut out a band and purify it.

Slab gels are cheap and simple but manual and coarse. Capillary electrophoresis, next, does the same separation with single-base resolution and automated fluorescent detection.

What to take from this

DNA runs to the anode and separates by size because charge scales with length. Pour agarose (0.7-3%) for most work and polyacrylamide for single-base resolution; use TAE or TBE. Size bands by interpolating against a ladder on a log scale. Read the controls first: a dimer in the NTC is harmless, a product band in the NTC means contamination, and a blank sample lane with a good ladder means the reaction failed.