Gel Electrophoresis
Related: PCR · Nucleic Acid Quantification · Sanger Sequencing · DNA Extraction
1. Principle
Agarose (or polyacrylamide) gel electrophoresis separates nucleic acids by size under an electric field; intercalating dyes make bands visible for confirming PCR products, integrity, or restriction patterns.
2. Step-by-Step Procedure (conceptual)
- Cast gel at % agarose matched to expected fragment sizes.
- Load samples with dye + DNA ladder.
- Run at appropriate voltage; image under blue/UV.
- Estimate size vs ladder; optional band excision for cloning/Sanger.
3. Interpretation
- Expected amplicon size supports specific PCR.
- Smearing → degradation or overload.
- Pitfalls: Ethidium UV damage; mis-sized bands from primer-dimers; contamination visible as extra bands.
4. Clinical Use Cases
- Teaching labs; troubleshooting NAATs; legacy RFLP.
- Largely replaced by real-time optics and capillary fragment analysis in modern clinical PCR — still valuable for QC and research.
5. Comparison with Other Methods
| Method | Readout |
|---|---|
| Gel | Size bands |
| qPCR | Fluorescence curves |
| Capillary electrophoresis | High-res fragments / Sanger |
6. Mnemonic / Visual Aid
Small fragments run farther — DNA is negatively charged toward the anode.
Active Recall
- Why change agarose percentage?
- What do primer-dimer bands look like?
- Why have clinical labs moved away from open-tube gel PCR?