In case of electrophoresis of single stranded DNA or RNA, which type of gels are used ? (a) Renaturing (b) Denaturing (c) The routine agarose gel (d) The routine polyacrylamide […]
Tag: Agarose Gel Electrophoresis
Agarose Gel / Agarose Gel Electrophoresis
Agarose Gel Electrophoresis (AGE) is a common molecular biology technique used to separate nucleic acids (DNA & RNA) based on their size (length in base pairs).
π What is Agarose?
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Agarose is a polysaccharide obtained from red algae (seaweed)
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It forms a porous, jelly-like matrix when heated and cooled in buffer
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The pore size depends on agarose concentration
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Higher % agarose β smaller pores β separates smaller DNA
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Lower % agarose β larger pores β separates bigger DNA
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β‘ Principle of Agarose Gel Electrophoresis
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DNA is negatively charged due to its phosphate backbone
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When an electric field is applied:
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DNA migrates from negative (cathode) β positive (anode)
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Smaller fragments move faster & farther, larger fragments move slowly
β‘ Separation is based on size (NOT charge)
π§ͺ Steps of Agarose Gel Electrophoresis
| Step | Process |
|---|---|
| 1 | Prepare agarose gel with buffer (TAE/TBE) |
| 2 | Add DNA stain (Ethidium Bromide / SYBR Safe) |
| 3 | Pour molten gel into casting tray with comb |
| 4 | Load DNA samples into wells |
| 5 | Run current through gel |
| 6 | Visualize bands under UV / Blue light |
π Interpretation
| Band position | Meaning |
|---|---|
| Band farther from well | Smaller fragment |
| Band closer to well | Larger fragment |
| Bright band | High concentration |
| Faint band | Low concentration |
Usually compared with DNA ladder / marker to determine fragment size.
π Concentration of Agarose Gel & Usage
| Agarose % | Fragment Size Range |
|---|---|
| 0.5% | 5 β 30 kb |
| 0.8% | 0.8 β 10 kb |
| 1.0% | 0.5 β 7 kb |
| 1.5% | 0.2 β 3 kb |
| 2.0% | 0.05 β 2 kb |
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