Experiment 03Electrophoresis • B.Sc. / M.Sc.
Horizontal Submarine Agarose Gel Electrophoresis (AGE) & DNA Fragment Sizing
🎯 Aim: To cast a $1.0% (text{w/v})$ agarose gel in $1timestext{TAE}$ buffer, load genomic and plasmid DNA samples with a calibrated $1,text{kb}$ DNA molecular ladder, separate fragments under a $5,text{V/cm}$ electric field, and visualize bands under UV transillumination using Ethidium Bromide.
🔬 Theoretical Principle & Sieving Physics
Agarose is a linear galactan polymer composed of alternating $(1to 3)$-linked $beta$-D-galactopyranose and $(1to 4)$-linked 3,6-anhydro-$alpha$-L-galactopyranose. When dissolved in boiling aqueous buffer and cooled below $40^circtext{C}$, agarose chains form coiled helical bundles that aggregate into a three-dimensional porous sieving network:
- Charge and Migration: Because of the repeating negative charges on phosphodiester backbones, DNA molecules possess a constant charge-to-mass ratio at neutral pH. In an electric field, all DNA fragments migrate toward the positive anode ($+$).
- Molecular Sieving: Migration velocity through the agarose matrix is inversely proportional to the $log_{10}$ of molecular weight ($mu propto 1/log_{10} M_r$). Smaller fragments slip easily through the pores, while large fragments experience high frictional resistance.
- Topological Mobility: For an identical plasmid, electrophoretic mobility follows:
$$text{Supercoiled Form I (ccc)} > text{Linear Form III} > text{Nicked Open Circular Form II (oc)}$$
Supercoiled DNA is tightly wound and compact, meeting minimum frictional drag. Nicked open circular DNA has a floppy, relaxed conformation that experiences maximum resistance. - Fluorescent Intercalation: Ethidium bromide (EtBr) inserts between stacked planar aromatic base pairs. Upon excitation by UV light at $lambda = 302,text{nm}$, energy is transferred to the dye, emitting orange-red fluorescence ($lambda_{em} = 590,text{nm}$) with an intensity directly proportional to DNA quantity.
Figure 3.1: Horizontal Submarine Agarose Gel Electrophoresis (AGE) & DNA Resolution
📋 Agarose Gel Casting Formulation Table
| Gel Concentration | Linear DNA Resolution Range | Recommended Application | Agarose in 50 mL 1× TAE |
|---|---|---|---|
| 0.7% (w/v) | $1.0,text{kb} – 15.0,text{kb}$ | Genomic DNA, Large BAC / Cosmids | 0.35 g |
| 1.0% (w/v) | $0.5,text{kb} – 8.0,text{kb}$ | Plasmids, Routine Restriction Digestion | 0.50 g |
| 1.5% (w/v) | $0.2,text{kb} – 3.0,text{kb}$ | PCR Amplicons, RT-PCR Products | 0.75 g |
| 2.0% (w/v) | $0.1,text{kb} – 1.5,text{kb}$ | Small PCR Amplicons, Restriction Fragments | 1.00 g |
💬 Viva-Voce Questions & Model Answers
Q1: What are the distinct roles of TAE and TBE running buffers?
TAE (Tris-Acetate-EDTA) provides optimal resolution for high-molecular-weight DNA fragments ($> 2,text{kb}$) and is preferred when extracting DNA bands for cloning because borate inhibits subsequent ligases. TBE (Tris-Borate-EDTA) has a higher buffering capacity and electrical resistance, making it superior for resolving small DNA fragments ($< 1,text{kb}$) and long electrophoretic runs.
Q2: What is the function of Bromophenol Blue and Xylene Cyanol in DNA loading dyes?
They serve as visual tracking dyes. In a 1% agarose gel, Bromophenol Blue co-migrates with linear DNA fragments of approximately $300–500,text{bp}$, while Xylene Cyanol FF co-migrates with fragments of $approx 4,text{kb}$, indicating when to terminate the electric run without exposing the gel to UV.
Q3: Why does voltage affect DNA resolution in agarose gel electrophoresis?
Operating above $5–8,text{V/cm}$ generates excessive Joule heating ($P = I^2R$), which decreases gel viscosity, broadens bands via thermal diffusion, causes bands to warp (“smiling bands”), and can melt low-melting-point agarose.