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    Chapter 8

    Uploaded by

    THỊNH HUỲNH LÊ ĐỨC

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    Chapter 8

    Uploaded by

    THỊNH HUỲNH LÊ ĐỨC
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    Chapter 8

    Uploaded by

    THỊNH HUỲNH LÊ ĐỨC

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    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
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    Chapter 8: Agarose Gel Electrophoresis Method

    Objectives

    After completing this lesson, learners will be able to:

    1. Prepare an agarose gel electrophoresis system.


    2. Analyze nucleotide sequences (PCR products, DNA, RNA) using agarose gel
    electrophoresis.

    8.1 Introduction

    Electrophoresis is a technique that uses an electric field to separate mixtures of DNA, RNA, or
    proteins on a three-dimensional network matrix. Nucleic acids, carrying a negative charge due to
    the phosphate groups in their molecular structure, migrate towards the anode in an electric field.
    The migration rate of nucleic acids through the matrix depends on their size and conformation.
    The matrices used in electrophoresis include paper, cellulose acetate, starch gel, … Among them,
    agarose gel and polyacrylamide gel are the most commonly used.

    Agarose and agaropectin are the two main components of agar, which is extracted from seaweed.
    Agarose is a linear polysaccharide composed of repeating disaccharide units of agarobiose. In
    powdered form, agarose becomes hydrated and dissolves in water when heated above 85°C.
    Upon cooling below 45°C, hydrogen bonds and electrostatic interactions between agarose
    molecules form, creating a gel with a network structure. Agarose gel is chemically and
    physically inert. The pore size of the gel varies depending on the concentration of agarose, which
    allows for the separation of nucleic acids of different sizes (Table 8.1).

    Electrophoresis on agarose gels is typically carried out horizontally, using a buffer system to
    provide ions for current conduction, maintain pH, and inhibit nucleases that could degrade
    nucleic acids during the run. The two most common buffer systems are TAE (Tris-acetate-
    EDTA) and TBE (Tris-borate-EDTA). TAE is preferred for larger DNA fragments due to its
    lower buffering capacity and minimal enzyme inhibition, whereas TBE, with its higher buffering
    capacity and better resolution for small nucleic acids (< 2 kb), is suited for longer electrophoresis
    runs. Consequently, TAE is often chosen for routine electrophoresis, while TBE is reserved for
    more demanding applications.

    Agarose gel electrophoresis is performed horizontally, with the gel submerged in running buffer,
    thus referred to as horizontal submerged electrophoresis (Figure 8.1). The running buffer serves
    to (1) provide ions to create a conductive environment; (2) stabilize pH to maintain the negative
    charge of nucleic acids; and (3) inhibit nucleases to prevent nucleic acid degradation during
    electrophoresis. The two common running buffers are TAE (Tris-acetate-EDTA) and TBE (Tris-
    borate-EDTA). TAE buffer is used for analyzing large DNA and purifying DNA for cloning, as
    borate in TBE can inhibit certain DNA-modifying enzymes. TBE buffer provides higher
    resolution for nucleic acids smaller than 2 kb and has better buffering capacity than TAE,
    making it suitable for longer electrophoresis runs.

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