Agarose gel electrophoresis
Separate DNA fragments by size to check PCR products, digests and integrity.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1How a DNA gel separates fragments
DNA carries one negative charge per phosphate, so every fragment has about the same charge per unit length. In an electric field all of it moves toward the positive electrode, and what separates fragments is the gel: agarose forms a mesh of pores, and longer molecules thread through it more slowly than short ones.
Large fragments
Big pores separate roughly 0.5–12 kb: plasmids, digests, genomic DNA integrity.
Medium fragments
Tighter pores resolve about 0.1–3 kb, including bands only tens of bp apart.
Small fragments
For products under ~500 bp. Large fragments barely leave the well.
2Reading the gel
A ladder of known sizes runs in one lane. Because migration distance is close to linear in log(size), you read a band's size by comparing it with the ladder bands above and below it. Brightness reflects mass: a band with twice the DNA glows about twice as bright, until the camera saturates.
3Loading dye, stain and voltage
- Loading dye adds glycerol or Ficoll so the sample sinks, and coloured dyes (bromophenol blue, xylene cyanol) that run at known apparent sizes so you can follow the run.
- The stain (SYBR Safe, GelRed or ethidium bromide) binds DNA and fluoresces under blue or UV light. It can be cast in the gel or used to post-stain.
- 5–8 V/cm (about 80–120 V on a mini-gel) gives sharp bands in 30–60 min. Much higher voltage heats the gel, which blurs and bends bands.
Key terms
Practise these as flashcards →A mix of DNA fragments of known sizes run alongside samples for sizing.
How fast a fragment moves per unit field; falls with log(size) and with gel percentage.
Twisted circular plasmid; runs faster than linear DNA of the same size.
Continuous signal down a lane: degraded DNA, overloading or a mix of many sizes.
Above ~20–50 kb all DNA moves at the same speed in a constant field, so sizes can't be separated without pulsed fields.
Running buffers. TAE suits large fragments and recovery; TBE gives sharper small bands and holds up in long runs.
When to use it
- Checking PCR products, restriction digests or plasmid preps for the expected size.
- Checking genomic DNA or RNA integrity before downstream steps.
- Purifying a fragment of the right size (gel extraction) before cloning.
Compared with alternatives
The usual source of samples: a gel confirms the PCR made one product of the right size.
Bioanalyzer/TapeStation give automated, quantitative sizing from tiny volumes; a gel is cheaper and shows more at once.
The protein equivalent: SDS gives proteins uniform charge so polyacrylamide separates them by mass.
Transfers the separated DNA to a membrane to detect one sequence with a probe.
How the gel is imaged and quantified.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Cut the band you want out of the gel and recover the DNA, usually on a silica spin column, to separate a product from off-target bands or primers before cloning.
- Separate the bands well first: at least ~0.5 cm between the band you want and its neighbours.
- Cut under blue light with SYBR-type stains; UV damages DNA within seconds.
- Trim the slice tightly: columns hold a limited amount of dissolved agarose.