You load four DNA samples into a 1% agarose gel: fragments of 200 bp, 800 bp, 2000 bp, and 5000 bp. After 45 minutes of electrophoresis, which fragment has traveled the greatest distance from the loading well?
A5000 bp — larger fragments carry more charge and are pulled harder by the electric field
B2000 bp — mid-sized fragments find the optimal balance between charge and mass
C800 bp — small fragments move faster through the gel matrix
D200 bp — smallest fragments face the least resistance in the gel matrix and migrate furthest
The most common misconception is that larger fragments migrate farther because they carry more charge. In reality, all DNA fragments have the same charge-to-mass ratio (one negative charge per phosphate, per nucleotide). Charge and mass scale together, so the net electrical force per unit mass is the same for all fragments. The determining factor is the gel matrix, which acts as a sieve: smaller fragments navigate the pores more easily and migrate farther. Option A reflects the classic misconception.
Question 2 Multiple Choice
SDS-PAGE requires denaturation of proteins with sodium dodecyl sulfate before electrophoresis. Why is this extra step necessary for proteins but not for DNA?
ADNA is more heat-stable than proteins and can withstand the electric field without denaturing
BProteins are too large to enter the gel matrix without unfolding
CDNA has a uniform charge-to-mass ratio due to its phosphate backbone; protein charge varies with amino acid composition, so without SDS they do not migrate by size alone
DSDS stains proteins so they can be visualized, similar to how ethidium bromide stains DNA
The key is the charge-to-mass ratio. Every nucleotide in DNA contributes one phosphate group with one negative charge, so charge scales directly with length — all DNA fragments migrate at the same charge-to-mass ratio, and size alone determines speed. Proteins, by contrast, have variable amino acid compositions: some are positively charged, some negatively charged, some near neutral at physiological pH. Without SDS, a highly charged small protein might migrate faster than a large less-charged protein, making size comparison meaningless. SDS denatures proteins into linear chains and coats them uniformly with negative charge proportional to their length, imposing the same uniform charge-to-mass ratio that DNA has naturally.
Question 3 True / False
A brighter band on an ethidium bromide-stained agarose gel indicates that there is more DNA of that particular fragment size in the sample.
TTrue
FFalse
Answer: True
Ethidium bromide (and modern safer alternatives like SYBR Safe) intercalates between stacked base pairs of double-stranded DNA. The more DNA molecules present, the more dye intercalates, and the brighter the fluorescence under UV light. Band intensity is therefore proportional to the mass (amount) of DNA at that size. This is useful for comparing DNA concentrations across lanes and for estimating yield — for example, comparing the intensity of a PCR product band to a known-quantity ladder fragment.
Question 4 True / False
In agarose gel electrophoresis of DNA, larger fragments migrate farther from the loading wells than smaller fragments during the same electrophoresis run.
TTrue
FFalse
Answer: False
Smaller DNA fragments migrate farther, not larger. The gel matrix acts as a molecular sieve: small molecules navigate the pores more easily and move faster. Larger fragments are impeded by the matrix and move more slowly, ending up closer to the loading wells after the same elapsed time. This inverse relationship between size and migration distance is what makes gel electrophoresis a size-separation technique.
Question 5 Short Answer
Why is the gel matrix essential for size-based separation of DNA? What would happen if you applied an electric field to DNA in free solution without a gel?
Think about your answer, then reveal below.
Model answer: In free solution, all DNA fragments have the same charge-to-mass ratio (constant per nucleotide). The electric force and the viscous drag would both scale with mass, so all fragments would migrate at the same velocity regardless of size — no separation would occur. The gel matrix creates size-dependent friction: it is a tangled polymer network whose pore sizes obstruct larger molecules more than smaller ones. This differential sieving is the only reason different-sized fragments separate into distinct bands.
This is why gel composition matters: a low-concentration gel (0.5–0.8%) has larger pores and separates large DNA fragments (5–50 kb) better; a high-concentration gel (2–3%) has smaller pores and resolves small fragments (50–500 bp) better. Choosing the right gel percentage for your expected fragment size range is a practical application of understanding the sieving mechanism.