AP Syllabus focus:
‘Gel electrophoresis separates DNA fragments based on size and charge, producing banding patterns that can be compared between samples.’
Gel electrophoresis is a foundational lab method for separating DNA into visible bands. Understanding how DNA’s charge, a gel matrix, and an electric field interact helps you interpret banding patterns and compare multiple samples reliably.
Principle of DNA separation
Gel electrophoresis: A technique that uses an electric field to move charged molecules through a porous gel, separating them primarily by size (and sometimes shape).
DNA fragments have a net negative charge because of phosphate groups in the sugar-phosphate backbone. When placed in an electric field, DNA migrates through the gel toward the positive electrode.
Why fragments separate
The gel (commonly agarose) acts like a molecular sieve.
Smaller DNA fragments navigate gel pores more easily and move farther/faster.
Larger fragments experience more resistance and move shorter/slower.
Because DNA has a relatively consistent charge-to-mass ratio, separation is dominated by fragment length (size).
Core components of a gel electrophoresis setup
Gel, buffer, and chamber
Gel matrix: Agarose is melted, poured into a tray, and allowed to solidify.
Comb and wells: A comb creates wells where DNA samples are loaded.
Running buffer: An ion-containing solution (e.g., TAE/TBE) that:
conducts electricity
maintains pH so DNA remains stable and charged
Electrodes and power supply: Create the electric field across the gel.
Samples and reference standards
DNA ladder (molecular weight marker): A mixture of DNA fragments of known sizes used to estimate the sizes of sample fragments by comparison.
A loading dye is mixed with DNA to help the sample sink into wells and to provide visible tracking dyes that approximate the progress of DNA migration.
Running the gel (process focus)
Careful, consistent technique is key because comparisons between samples depend on equal conditions across lanes.
Prepare agarose gel at an appropriate concentration for the expected fragment sizes.
Place the gel in the chamber and cover it with running buffer.
Load samples into wells:
include a DNA ladder
load similar volumes to support valid comparisons
Apply voltage:
DNA migrates from the negative end (near wells) toward the positive end
fragments begin separating into distinct bands
Stop the run when separation is sufficient (bands spread out without running off the gel).
Visualising and interpreting banding patterns
DNA is not inherently visible, so gels are stained with a DNA-binding dye (commonly a fluorescent stain) and viewed under an appropriate light source. Separated DNA appears as bands in lanes.
What a “banding pattern” means
Each band represents many DNA molecules of the same length grouped together.
Band position indicates relative size:
farther from the wells typically means smaller fragments
Band intensity reflects relative amount of DNA:
brighter/thicker bands usually indicate more DNA
Comparing samples (syllabus emphasis)
Banding patterns can be compared between samples by examining:
presence/absence of bands at similar positions
relative spacing between bands
correspondence with the DNA ladder to infer approximate fragment sizes
Valid comparisons require that samples were run on the same gel (or under identical conditions) with the same ladder and similar loading volumes.
Factors that affect separation quality
Gel concentration: Higher agarose slows migration and can improve separation of smaller fragments; lower agarose helps resolve larger fragments.
Voltage and run time: Excessive voltage can overheat the gel, distort bands, or reduce resolution.
Sample quality: Degraded DNA can produce smears rather than discrete bands.
Overloading wells: Too much DNA can cause thick, merged, or streaky bands.
Uneven gel/wells: Crooked wells or bubbles can cause distorted migration and unreliable lane comparisons.
FAQ
DNA conformation can affect movement. Supercoiled, linear, and nicked circular DNA of identical base-pair length can migrate at different rates through the gel matrix.
Match pore size to fragment range. Higher % agarose resolves smaller fragments; lower % resolves larger fragments. Many labs use mid-range gels when the expected sizes are broad.
Uneven heating and electric field strength can make outer lanes run differently from the centre. This is often linked to high voltage, low buffer volume, or poor heat dissipation.
Common causes include degraded DNA, contaminants (e.g., salts), overloading the well, or running the gel too long so fragments spread out and lose sharp boundaries.
Yes. A target band can be excised and purified using gel extraction methods that dissolve agarose and bind DNA to a column or matrix, allowing elution into clean buffer.
Practice Questions
Explain why DNA fragments move towards the positive electrode during gel electrophoresis and why smaller fragments travel further than larger fragments. (2 marks)
DNA has a net negative charge due to phosphate groups and is attracted to the positive electrode (1).
The gel acts as a sieve; smaller fragments pass through pores more easily and migrate further/faster (1).
Describe how gel electrophoresis can be used to compare DNA samples using banding patterns. Include the role of a DNA ladder and two factors that must be controlled to make the comparison valid. (5 marks)
DNA fragments are separated into bands according to size as they migrate through the gel (1).
Banding patterns are compared by matching band positions between lanes (presence/absence and relative positions) (1).
A DNA ladder provides known fragment sizes to estimate sample fragment sizes by comparison (1).
Control factor: same gel/buffer/voltage/run time so migration distances are comparable (1).
Control factor: similar sample loading volume/concentration to avoid misleading band intensity or smearing (1).
