Polyacrylamide Gel Electrophoresis

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Questions and Answers

What is the purpose of a gel in gel electrophoresis?

To help conduct electricity through ions

To provide structural support to the samples

To separate molecules based on size (correct)

To speed up the movement of molecules

Which component of the gel electrophoresis system conducts electricity by running buffer?

Voltage power supply

Buffer system (correct)

Supporting medium

Agarose gel

What is the main difference between vertical and horizontal gel electrophoresis setups?

The type of gel used

Speed of the separation process

Conductivity of the buffer system

Orientation of the supporting medium (correct)

Which type of gel is primarily used for separating proteins and nucleic acids in gel electrophoresis?

Polyacrylamide gel Signup and view all the answers

What distinguishes agarose gel from polyacrylamide gel in terms of usage in electrophoresis?

Agarose gel is used for proteins, polyacrylamide for nucleic acids Signup and view all the answers

What is the primary advantage of polyacrylamide gel electrophoresis over agarose gel electrophoresis?

It has a higher resolving power for separating molecules. Signup and view all the answers

What is the primary advantage of agarose gel electrophoresis over polyacrylamide gel electrophoresis?

Agarose gels are more effective for separating larger DNA fragments. Signup and view all the answers

What is the primary reason for the gelling property of agarose?

Inter- and intra-molecular hydrogen bonding within agarose molecules. Signup and view all the answers

What is the primary advantage of using polyacrylamide gels over agarose gels for DNA separation?

Polyacrylamide gels have a higher resolving power for separating small DNA fragments. Signup and view all the answers

What is the primary component of agarose?

Repeating units of agarobiose, consisting of alternating galactose and 3,6-anhydrogalactose Signup and view all the answers

What is the primary principle behind agarose gel electrophoresis?

Separation of molecules based on their rate of movement through the gel under an electric field. Signup and view all the answers

Which of the following is an advantage of agarose gel electrophoresis?

Easy to prepare and requires a small concentration of agarose Signup and view all the answers

Which of the following statements about polyacrylamide gel electrophoresis (PAGE) is correct?

Staining can be done before pouring the gel Signup and view all the answers

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?

The difference in their conformations Signup and view all the answers

Which of the following statements about agarose is incorrect?

It provides better resolution than polyacrylamide gels Signup and view all the answers

Which of the following factors does not determine the migration rate of a molecule through a gel?

The base composition of the molecule Signup and view all the answers

What is the purpose of using topoisomerases or introducing a nick in one of the DNA strands during gel electrophoresis?

To separate supercoiled circular DNA into relaxed circular DNA Signup and view all the answers

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.