Electrophoresis Flashcards
What is electrophoresis?
The movement of molecules by an electric current in air, solution, or a gel matrix.
Why do DNA and RNA migrate toward the positive pole (anode)?
Because each phosphate group is negatively charged.
How does molecular size affect migration speed in gel electrophoresis?
Smaller molecules move faster, while larger molecules move slower.
What concentration of agarose gel is best for small DNA fragments (50–500 bp)?
2%–3% agarose gel
What concentration of agarose gel is best for large DNA fragments (2,000–50,000 bp)?
0.5%–1% agarose gel
What affects agarose gel strength over time?
Chaotropic agents like urea and prolonged use.
What makes polyacrylamide gels different from agarose gels?
They are synthetic, allowing precise control of polymer properties.
What is polyacrylamide gel commonly used for?
High-resolution applications like:
• DNA sequencing
• Mutation analysis
• Nuclease protection assays
What is a key advantage of capillary electrophoresis?
It provides rapid, automated, and high-resolution separation of molecules.
How does capillary electrophoresis separate particles?
By size (small = fast, large = slow) and charge (negative = fast, positive = slow).
Why is a polymer (gel) used in capillary electrophoresis?
It resolves DNA fragments mostly by size since DNA’s charge and size oppose each other.
How do molecules behave at different pH levels in capillary electrophoresis?
• High pH: Negatively charged molecules are completely ionized.
• Low pH: Positively charged molecules are completely protonated.
What is the purpose of buffer systems in electrophoresis?
To carry the current and protect the samples.
What is the most commonly used buffer in electrophoresis?
Tris-buffer.
What is the function of bromophenol blue?
It is a tracking dye that helps monitor migration but does not bind to DNA.
How does ethidium bromide (EtBr) help visualize DNA?
It intercalates DNA and emits orange fluorescence (300 nm) under UV light.
How does SYBR Green differ from EtBr?
It does not intercalate DNA but binds in the minor groove, emitting 522 nm (orange range) light.
What is the purpose of silver staining in electrophoresis?
It was originally for protein visualization, but it can also detect DNA and RNA.
What are the steps of silver staining?
- Fixation with methanol and acetic acid.
- Impregnation with ammoniacal silver or silver nitrate in acidic solution.
- Silver ion interaction with nucleophilic groups.
- Crystallization of metallic silver with formaldehyde in acidic or alkaline solution.
What is the first step in silver staining?
Fixation – The sample is fixed with methanol and acetic acid to prevent diffusion
What happens after fixation in silver staining?
Impregnation – The gel is treated with ammoniacal silver (silver diamine) or silver nitrate in a weakly acidic solution.
What occurs when silver ions interact with nucleophilic groups in silver staining?
Silver ions bind to nucleophilic sites on DNA, RNA, or proteins, forming a latent image.
What is the final step in silver staining?
Development – Silver ions are reduced to metallic silver, forming a black precipitate when exposed to formaldehyde in acidic or alkaline solution.
