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Questions and Answers
What is the purpose of a gel in gel electrophoresis?
Which component of the gel electrophoresis system conducts electricity by running buffer?
What is the main difference between vertical and horizontal gel electrophoresis setups?
Which type of gel is primarily used for separating proteins and nucleic acids in gel electrophoresis?
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What distinguishes agarose gel from polyacrylamide gel in terms of usage in electrophoresis?
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What is the primary advantage of polyacrylamide gel electrophoresis over agarose gel electrophoresis?
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What is the primary advantage of agarose gel electrophoresis over polyacrylamide gel electrophoresis?
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What is the primary reason for the gelling property of agarose?
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What is the primary advantage of using polyacrylamide gels over agarose gels for DNA separation?
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What is the primary component of agarose?
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What is the primary principle behind agarose gel electrophoresis?
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Which of the following is an advantage of agarose gel electrophoresis?
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Which of the following statements about polyacrylamide gel electrophoresis (PAGE) is correct?
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What is the primary reason for the difference in migration rates of supercoiled circular DNA, relaxed circular DNA, and linear DNA of the same molecular weight through a gel?
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Which of the following statements about agarose is incorrect?
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Which of the following factors does not determine the migration rate of a molecule through a gel?
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What is the purpose of using topoisomerases or introducing a nick in one of the DNA strands during gel electrophoresis?
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Study Notes
DNA Gel Electrophoresis
- Developed in the 1970s by Phil Sharp, Joe Sambook, and Bill Sugden at Cold Spring Harbor Laboratory
- Uses an agarose gel, made from highly purified seaweed
Main Components of Gel Electrophoresis
- Voltage: Power supply
- Supporting medium: Paper, cellulose acetate, agarose gel, polyacrylamide gel
- Buffer system: Conducts electricity by running buffer (contains ions that allows for the conduction of electricity)
Gel Electrophoresis
- A type of electrophoresis in which supporting medium is a gel
- Separation is brought about through molecular sieving technique, based on molecular size of the substance
- Gel material acts as a “molecular sieve”
Types of Gel Electrophoresis Set-ups
- Vertical Gel Electrophoresis (GE): Supporting medium is vertical
- Horizontal Gel Electrophoresis (GE): Supporting medium is horizontal
Agarose Gel
- Used for nucleic acids
- DNA conformation affects migration rate: supercoiled circular DNA, relaxed circular DNA, and linear DNA of the same molecular weight will migrate at different rates through the gel
- Evaluation of DNA quality: a compact, no double bond or faint band indicates good quality
Advantages of Agarose Gel
- Easy to prepare and small concentration of agar is required
- Resolution is superior to that of filter paper
- Large quantities of proteins can be separated and recovered
- Adsorption of negatively charged protein molecule is negligible
- It absorbs proteins relatively less when compared to other medium
Disadvantages of Agarose Gel
- Resolution is less compared to PAGE
- Different sources and batches of agar tend to give different results and purification is often necessary
Agarose and PAGE
- Agarose: a polysaccharide extracted from seaweed, commonly used for DNA separations
- PAGE: a cross-linked polymer of acrylamide, used for DNA or protein separations
Factors that Determine Migration through a Gel
- Greater resolving power focuses on the separation of the electrophoretic system
- Molecular size and shape of the substance
- Pore size of the gel
- Voltage and buffer composition
Agarose Gel Properties
- Most effective in separating DNA fragments of varying sizes from 100 bp to 25kbp
- Agarose gels are formed by suspending dry agarose in aqueous buffer, heating until clear solution is obtained, and cooling the solution to room temperature
- Extracted from seaweed, a linear carbohydrate polymer composed of repeating units of agarobiose
- The gelling property is due to both inter- and intra- molecular hydrogen bonding and not due to polymerization
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Description
Learn about the features and advantages of using polyacrylamide gel electrophoresis for DNA separation. Understand how this technique allows for greater resolving power, accommodating larger DNA samples, and recovery of extremely pure DNA.