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    Southern Blotting Technique

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    SouravS.Panda
    The document describes the Southern blotting technique. [1] Southern blotting allows detection of a specific DNA sequence in a complex DNA sample. [2] It involves separating DNA fragments by size using agarose gel electrophoresis, transferring the DNA to a membrane, and detecting a labeled DNA probe that is complementary to the sequence of interest. [3] Southern blotting has applications in gene discovery, genetic mapping, and diagnostics.

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    Southern Blotting Technique

    Uploaded by

    SouravS.Panda
    242 views15 pages
    The document describes the Southern blotting technique. [1] Southern blotting allows detection of a specific DNA sequence in a complex DNA sample. [2] It involves separating DNA fragments by size using agarose gel electrophoresis, transferring the DNA to a membrane, and detecting a labeled DNA probe that is complementary to the sequence of interest. [3] Southern blotting has applications in gene discovery, genetic mapping, and diagnostics.

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    southern blotting technique

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    The document describes the Southern blotting technique. [1] Southern blotting allows detection of a specific DNA sequence in a complex DNA sample. [2] It involves separating DNA fragments by size using agarose gel electrophoresis, transferring the DNA to a membrane, and detecting a labeled DNA probe that is complementary to the sequence of interest. [3] Southern blotting has applications in gene discovery, genetic mapping, and diagnostics.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    242 views15 pages

    Southern Blotting Technique

    Uploaded by

    SouravS.Panda
    The document describes the Southern blotting technique. [1] Southern blotting allows detection of a specific DNA sequence in a complex DNA sample. [2] It involves separating DNA fragments by size using agarose gel electrophoresis, transferring the DNA to a membrane, and detecting a labeled DNA probe that is complementary to the sequence of interest. [3] Southern blotting has applications in gene discovery, genetic mapping, and diagnostics.

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    VRIJE UNIVERSITEIT BRUSSELS

    INTERUNIVERSITY PROGRAMME MOLECULAR BIOLOGY

    GENERAL PRACTICAL
    COURSE

    SOUTHERN BLOTTING
    TECHNIQUE
    STUDENT: BUI PHUONG THAO
    COURSE : IPMB 2010-2012

    SOUTHERN BLOT
    I. INTRODUCTION

    II. PRINCIPLE

    III. PROTOCOL

    IV. APPLICATIONS

    I. INTRODUCTION
    A technique which
    allows the detection of a
    specific DNA sequence
    (gene or other) in a large,
    complex sample of DNA
    (e.g. cellular DNA).

    Named
    after
    its
    inventor and developer,
    the British biologist Edwin
    M. Southern in 1975.

    II. PRINCIPLE
    Southern blotting combines agarose gel
    electrophoresis for size separation of DNA with
    methods to transfer the size-separated DNA to a
    filter membrane for probe hybridization.

    III. PROTOCOL
    Step 1
    DNA separation

    Step 2
    Blot on membrane

    Step 3
    Label with specific DNA probe

    Step 4
    Detect probe

    Step 1: DNA separation


    1

    DNA is
    extracted
    from the
    cells and
    purified

    DNA is
    restricted
    with
    enzymes

    A large
    piece of
    DNA is
    chopped
    into smaller
    pieces
    using RE

    2 3

    Loading and running


    DNA agarose gel
    Lane 1 DNA marker
    Lane 2 - Restricted DNA
    Lane 3 - Unrestricted (whole) DNA

    Step 2: Blot on membrane


    Denature double-stranded DNA into single strands by incubation with NaOH
    Transfer DNA to membrane

    20X SSC
    buffer

    Nylon or
    Nitrocellulose

    1216 hours

    Capillary flow method

    Step 2: Blot on membrane (cont)


    Two other methods for transferring DNA to a membrane

    Vacuum blotting
    - Quite fast (1-3 hours)
    - Gives excellent recoveries

    Electroblotting
    - More popular for polyacrylamide
    gels

    Step 3: Label with specific DNA probe


    Attach the transferred DNA to the membrane by high temperature or UV

    Incubate the blot with specific probe

    * A probe is a small (25-2000 bp) length of DNA or RNA


    - Complementary to the sequence (gene) of interest
    - Labeled by incorporating radioactivity or tagging with a fluorescent or
    chromogenic dye

    Step 4: Detect probe


    Unbound probe is washed away
    from the membrane
    (in 2X SSC buffer + 0.1% SDS)

    Detect the pattern of hybridization

    By autoradiography
    on X-ray film (in
    case of radioactive
    or fluorescent
    probe)

    By development of
    color on the
    membrane (in case
    of chromogenic
    probe)

    Example

    Figure. The figure on the left shows a photograph of a 0.7% agarose gel that has 14 different samples
    loaded on it. Each sample of DNA has been digested with the same restriction enzyme (EcoRI). The DNA
    was transferred to nitrocellulose and then probed with a radioactive fragment of DNA that was derived
    from the transformed gene. The figure on the right is a copy of the X-ray film and reveals which strains
    contain the target DNA and which ones do not.

    SUMMARY OF SOUTHERN BLOT PROTOCOL

    IV. APPLICATION
    SB is used in gene discovery and mapping, evolution and
    development studies, diagnostics and forensics:
    Determine the molecular weight of a restriction fragment
    and to measure relative amounts in different samples
    Detect the presence of a particular bit of DNA in a sample

    Analyze the genetic patterns which appear in a person's


    DNA
    Analyze restriction digestion fragmentation of DNA or a
    biological sample
    In regards to genetically modified organisms

    Detect RFLP and VNTRs

    References
    http://en.wikipedia.org/wiki/Southern_blot
    http://www.bio.davidson.edu/courses/genomics/.../Southernblot.html
    http://www.drylab1.vet.cornell.edu/Block4/Reference/Diagnostics/sout
    h.html
    http://www.molecularstation.com/dna/southern-blot/
    http://www.gbiosciences.com/EducationalProducts/Southern-BlotAnalysis.aspx
    http://www.koreanbio.org/Biocourse/index.php/Hybridization
    http://www.nationaldiagnostics.com/article_info.php/articles_id/72
    http://www.web-books.com/MoBio/Free/Ch9D.htm
    http://www.abe.leeward.hawaii.edu/WebSharing/Southern_Melzer.ppt

    THANK YOU FOR ATTENTION !

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