Podcast
Questions and Answers
What is the role of electroosmotic flow in capillary electrophoresis?
What is the role of electroosmotic flow in capillary electrophoresis?
- To slow down the migration of cations
- To increase friction in the capillary
- To move fluid toward the negative electrode (correct)
- To neutralize negative charges on the capillary wall
Electroosmotic flow cannot occur without understanding its underlying theory.
Electroosmotic flow cannot occur without understanding its underlying theory.
False (B)
What happens when high voltage is applied during electrophoresis?
What happens when high voltage is applied during electrophoresis?
The DNA band can turn into a parabolic line due to friction.
The inner surface of the capillary is coated with __________ charged groups.
The inner surface of the capillary is coated with __________ charged groups.
Match the type of mobility with its corresponding characteristic:
Match the type of mobility with its corresponding characteristic:
What is one advantage of capillary electrophoresis over conventional electrophoresis?
What is one advantage of capillary electrophoresis over conventional electrophoresis?
Cations in the diffuse layer are rigidly bound to the capillary wall.
Cations in the diffuse layer are rigidly bound to the capillary wall.
What type of information does capillary electrophoresis yield?
What type of information does capillary electrophoresis yield?
What is the primary function of loading dye in DNA electrophoresis?
What is the primary function of loading dye in DNA electrophoresis?
Ethidium bromide makes DNA visible under UV light by fluorescing orange.
Ethidium bromide makes DNA visible under UV light by fluorescing orange.
What is a DNA ladder used for during electrophoresis?
What is a DNA ladder used for during electrophoresis?
The process of separating charged molecules by their charge to mass ratio in a capillary tube is known as __________.
The process of separating charged molecules by their charge to mass ratio in a capillary tube is known as __________.
Match the following components involved in DNA electrophoresis to their functions:
Match the following components involved in DNA electrophoresis to their functions:
Which of the following is NOT a safer alternative to ethidium bromide for staining DNA?
Which of the following is NOT a safer alternative to ethidium bromide for staining DNA?
The DNA samples are allowed to fully migrate out of the gel before the electrophoresis is stopped.
The DNA samples are allowed to fully migrate out of the gel before the electrophoresis is stopped.
What does the term 'electrophoretic front' refer to?
What does the term 'electrophoretic front' refer to?
What factor primarily influences the speed at which charged macromolecules migrate in electrophoresis?
What factor primarily influences the speed at which charged macromolecules migrate in electrophoresis?
Supercoiled plasmids migrate slower than linearized plasmids during electrophoresis.
Supercoiled plasmids migrate slower than linearized plasmids during electrophoresis.
What is the role of molecular weight markers in gel electrophoresis?
What is the role of molecular weight markers in gel electrophoresis?
In agarose gel electrophoresis, DNA fragments migrate towards the _______ charged anode.
In agarose gel electrophoresis, DNA fragments migrate towards the _______ charged anode.
Match the type of electrophoresis with its commonly used matrix:
Match the type of electrophoresis with its commonly used matrix:
Which of the following factors does NOT affect electrophoretic mobility?
Which of the following factors does NOT affect electrophoretic mobility?
Agarose gel is commonly used for separating protein fragments in electrophoresis.
Agarose gel is commonly used for separating protein fragments in electrophoresis.
What happens to shorter DNA fragments in agarose gel electrophoresis?
What happens to shorter DNA fragments in agarose gel electrophoresis?
Flashcards
Electrophoresis
Electrophoresis
A laboratory technique that separates charged molecules based on size, charge, and shape using an electric field.
Electrophoretic Mobility
Electrophoretic Mobility
The movement of a charged molecule in an electric field. It's faster for molecules with a higher charge.
Support Matrix
Support Matrix
The material that acts as a sieve in electrophoresis, allowing molecules to migrate at different rates.
Agarose Gel
Agarose Gel
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DNA Fragments
DNA Fragments
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Distance Migrated
Distance Migrated
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Molecular Weight Markers
Molecular Weight Markers
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Agarose Gel Electrophoresis
Agarose Gel Electrophoresis
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Agarose Buffer
Agarose Buffer
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Loading Dye
Loading Dye
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DNA Ladder
DNA Ladder
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Gel Electrophoresis
Gel Electrophoresis
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Ethidium Bromide
Ethidium Bromide
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Capillary Electrophoresis
Capillary Electrophoresis
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Automated DNA Sequencing
Automated DNA Sequencing
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What is Electroosmotic Flow (EOF)?
What is Electroosmotic Flow (EOF)?
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How does EOF work?
How does EOF work?
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How does DNA size affect its movement in capillary electrophoresis?
How does DNA size affect its movement in capillary electrophoresis?
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What causes the 'smiley gel' effect?
What causes the 'smiley gel' effect?
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What are the benefits of Capillary Electrophoresis?
What are the benefits of Capillary Electrophoresis?
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Study Notes
Electrophoresis Overview
- Electrophoresis is a technique used to separate charged macromolecules in an electric field. Molecules are separated based on size, charge, and conformation.
- Charged molecules migrate toward either the positive or negative pole depending on their charge.
Factors Affecting Electrophoresis
- The greater the charge on a molecule, the faster it migrates.
- Larger molecules experience more friction and electrostatic forces, resulting in slower migration.
- Rounded molecules have less frictional/electrostatic retardation than non-globular ones.
- Supercoiled plasmids migrate faster than linearized plasmids.
Agarose Gel Electrophoresis (DNA)
- Agarose gel is used to separate DNA fragments based on their molecular weight.
- DNA (and RNA) molecules are negatively charged.
- In an electric field, they migrate to the positive electrode.
- DNA fragments are separated by size due to a consistent mass/charge ratio in agarose gels.
- Shorter DNA fragments move faster than longer ones.
- Distance migrated correlates inversely with the logarithm of molecular weight.
How to Cast an Agarose Gel
- Prepare a gel tray.
- Boil agarose powder in buffer.
- Pour the molten agarose into the tray.
- Insert a comb.
- Allow the gel to set.
Loading DNA into Wells
- Mix DNA samples with loading dye.
- Loading dye (containing glycerol/sucrose and Bromophenol Blue) helps the DNA settle to the bottom of the well.
- Bromophenol Blue acts as a marker to track migration.
- Load the samples into the wells of the gel.
DNA Ladder
- A DNA molecular weight marker comprised of DNA standards with known base pair sizes.
- Used to determine the approximate size of DNA samples.
- Run the ladder alongside the DNA samples on the gel.
Staining DNA in Agarose Gel
- DNA is invisible to the naked eye.
- Ethidium bromide binds to double-stranded DNA and fluoresces under UV light (orange).
- Ethidium bromide is often added to the gel or buffer before electrophoresis.
- After electrophoresis, the gel is visualized under UV light.
- Safer alternatives, like crystal violet, methylene blue, or propidium iodide, are available.
Capillary Electrophoresis
- A method to separate charged molecules based on their charge-to-mass ratio using a capillary tube in an electric field.
- Combines electrophoresis (movement in an electric field) and chromatography (measuring the time to travel a matrix).
- Automated DNA sequencing often uses capillary electrophoresis.
Electroosmotic Flow (EOF)
- The force that moves fluid through a capillary tube.
- The capillary tube's inner surface is typically coated with negative charges.
- This attracts positively charged ions to the tube walls (creating a diffuse layer).
- Applying a voltage causes EOF.
Capillary Electrophoresis Advantages
- High speed due to high voltage.
- High resolution, even separating DNA with a single base pair difference.
- Precise quantitative information using detectors and software.
- Requires very small sample volumes.
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Description
This quiz covers the fundamentals of electrophoresis, including the factors that affect the separation of charged molecules in an electric field. It specifically focuses on agarose gel electrophoresis and how it is utilized to separate DNA fragments based on size. Test your knowledge on the principles and applications of this essential laboratory technique.
