Explain gel electrophoresis

The Core Principle

Why does this work?

1. DNA is negatively charged: The phosphate groups in the sugar-phosphate backbone carry negative charges at neutral pH
2. Electric field creates force: When voltage is applied, DNA experiences electrostatic force toward the positive pole
3. Gel acts as molecular sieve: Agarose or polyacrylamide gel has pores; small DNA squeezes through easily, large DNA gets tangled and slowed

Step-by-Step: How It Works

1. Gel Preparation

What: Pour molten agarose gel (0.7–2% concentration) into a casting tray with a comb Why: Agarose polymerizes into a mesh network. The comb creates wells to load samples. Lower % = bigger pores (for large DNA), higher % = smaller pores (for small DNA) How: Heat agarose powder in buffer (TAE or TBE) until dissolved, cool slightly, pour, let solidify

2. Sample Loading

What: Mix DNA samples with loading dye (glycerol + tracking dye), pipette into wells Why: Loading dye makes samples dense (sink into wells) and visible (tracking dye shows how far the run has progressed) How: Typically load 5–20 μL per well; include a DNA ladder (molecular weight markers) in one well for size reference

3. Electrophoresis Run

What: Submerge gel in buffer (TAE/TBE), apply voltage (50–150 V) for 30–90 min Why: Buffer conducts electricity and maintains pH. DNA migrates from negative cathode toward positive anode How: DNA migration distance decreases linearly with the log of fragment size — i.e. $d = c - k\log(L)$

Mobility (correct definition): $\mu = \frac{v}{E} = \frac{d/t}{V/\ell}$ where $\mu$ is electrophoretic mobility (cm²·V⁻¹·s⁻¹), $v = d/t$ is the migration velocity, $E = V/\ell$ is the electric field (voltage $V$ divided by inter-electrode/gel length $\ell$). Defining mobility per unit field (not per unit voltage) makes it dimensionally correct and independent of the specific apparatus size.

Why voltage matters: Higher field = faster migration BUT too high causes heat → gel melting and band distortion. Typical: 5–10 V/cm (voltage divided by gel/electrode length).

4. Visualization

What: Stain gel with ethidium bromide (EtBr) or safer alternatives (SYBR Green), view under UV light Why: EtBr intercalates between DNA bases and fluoresces orange under UV (λ ~590 nm when excited at ~302 nm) How: Soak gel in stain solution 15–30 min, rinse, photograph on UV transilluminator

Key Variables & Optimization

Why TAE vs TBE? TBE has higher buffering capacity (resists pH change) → sharper bands for small DNA (<1000 bp). TAE is cheaper, better for large DNA (>1 kb) and DNA recovery.

Active Recall Checks

Mathematical Derivation: Why d Is Linear in log(L)

From first principles — the key is the reptation (snake-through) model of DNA moving in a gel.

1. Free-solution driving force: In free solution the electric force $q E$ (with charge $q \propto L$) and the hydrodynamic drag both scale with $L$, so mobility is size-independent — DNA of all sizes would move together. This is exactly why you need a gel to separate them.

2. Gel sieving via reptation: In a gel, a long DNA molecule must worm head-first through the pore network like a snake through a tube. Ogston/reptation theory shows that the fraction of gel pores a molecule can enter falls off roughly exponentially with its size: $\mu(L) \;\approx\; \mu_0 \, e^{-\beta L}$ for the low-field, size-fractionating regime, where $\beta$ depends on gel concentration and pore geometry.

3. Velocity and distance: With $v = \mu E$ and $d = v t = \mu_0 E t\, e^{-\beta L}$. Over the narrow window of $L$ where a given gel resolves well, this exponential is well-approximated (empirically and to first order) by a curve whose calibration plot of migration distance against $\log L$ is a straight line: $\boxed{\,d \;=\; c - k \log L\,}$

4. Why the empirical log-linear fit works: Between the gel's exclusion limit (very large DNA, all stuck near the well) and its resolution floor (very small DNA, all near the front), the response is smoothly monotonic. Plotting $\log L$ vs. distance linearizes this working range, which is why DNA ladders are read off a straight standard curve — the practical justification, rather than a claim that reptation is exactly log-linear everywhere.

Physical intuition: Small DNA slips through pores easily and races to the front; large DNA reptates slowly; the calibration is engineered to be a straight line in $\log L$ across the useful separation range.

Applications in Genetic Engineering

1. CRISPR verification: After Cas9 cuts, run gel to confirm target DNA cleaved into expected sizes
2. Cloning: Check insert ligated into vector (shift in plasmid size)
3. PCR genotyping: Different alleles give different band patterns
4. DNA fingerprinting: Restriction fragment length polymorphism (RFLP) analysis
5. Quality control: Assess DNA integrity before sequencing (sharp high-MW band = good)

Connections

• Restriction Enzymes – generate DNA fragments to analyze
• PCR – amplify DNA before running on gel
• Southern Blotting – next step after electrophoresis for specific sequence detection
• DNA Structure – why DNA is negatively charged (phosphate backbone)
• CRISPR-Cas9 Mechanism – gel verifies successful target cutting
• Plasmid Vectors – gel checks plasmid size changes after cloning
• DNA Sequencing Preparation – gel purification of correct-size fragments

Flashcards

#flashcards/biology

Why does DNA migrate toward the positive electrode in gel electrophoresis? :: DNA has a negatively charged sugar-phosphate backbone (phosphate groups are PO₄⁻) at neutral pH, so it is attracted to the positive anode by electrostatic force.

What determines how fast a DNA fragment moves through the gel? :: Size (molecular weight) – smaller fragments navigate through gel pores faster than larger fragments. Also affected by gel concentration, field strength, and buffer.

Concept Map

Hinglish (regional understanding)