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    Southern Blotting

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    Bisnis Daus
    The Southern blotting technique involves extracting DNA from cells, cutting the DNA into fragments using restriction enzymes, separating the fragments by size using gel electrophoresis, transferring the DNA fragments to a membrane, and detecting specific fragments by hybridizing labeled probes to the membrane-bound DNA and detecting the bound probes. The key steps are separating DNA fragments by size, immobilizing them on a membrane, hybridizing probes to detect specific fragments, and washing unbound probe away.

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    Southern Blotting

    Uploaded by

    Bisnis Daus
    41 views15 pages
    The Southern blotting technique involves extracting DNA from cells, cutting the DNA into fragments using restriction enzymes, separating the fragments by size using gel electrophoresis, transferring the DNA fragments to a membrane, and detecting specific fragments by hybridizing labeled probes to the membrane-bound DNA and detecting the bound probes. The key steps are separating DNA fragments by size, immobilizing them on a membrane, hybridizing probes to detect specific fragments, and washing unbound probe away.

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    The Southern blotting technique involves extracting DNA from cells, cutting the DNA into fragments using restriction enzymes, separating the fragments by size using gel electrophoresis, transferring the DNA fragments to a membrane, and detecting specific fragments by hybridizing labeled probes to the membrane-bound DNA and detecting the bound probes. The key steps are separating DNA fragments by size, immobilizing them on a membrane, hybridizing probes to detect specific fragments, and washing unbound probe away.

    Copyright:

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

    Southern Blotting

    Uploaded by

    Bisnis Daus
    The Southern blotting technique involves extracting DNA from cells, cutting the DNA into fragments using restriction enzymes, separating the fragments by size using gel electrophoresis, transferring the DNA fragments to a membrane, and detecting specific fragments by hybridizing labeled probes to the membrane-bound DNA and detecting the bound probes. The key steps are separating DNA fragments by size, immobilizing them on a membrane, hybridizing probes to detect specific fragments, and washing unbound probe away.

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    SOUTHERN

    BLOTTING
    Presented by:
    Imran Fakhroni
    Nurkholis Nugroho
    Nino Radiansyah
    Indra Ardi Fauzi
    Arfita Sari
    SOUTHERN BLOTTING
     The technique was developed by E.M.
    Southern in 1975.
     The Southern blot is used to detect the
    presence of a particular piece of DNA in a
    sample.
     The DNA detected can be a single gene, or
    it can be part of a larger piece of DNA such
    as a viral genome.
     The key to this method is hybridization.
     Hybridization-process of forming a double-
    stranded DNA molecule between a single-
    stranded DNA probe and a single-stranded
    target patient DNA.
    More..
    Steps for hybridization

    1. The mixture of molecules is separated.


    2. The molecules are immobilized on a matrix.
    3. The probe is added to the matrix to bind to the
    molecules.
    4. Any unbound probes are then removed.
    5. The place where the probe is connected
    corresponds to the location of the immobilized
    target molecule.
    I. DNA Purification

     Isolate the DNA in question from the rest


    of the cellular material in the nucleus.
     Incubate specimen with detergent to
    promote cell lysis.
     Lysis frees cellular proteins and DNA.
     Proteins are enzymatically degraded by
    incubation with proteinase.
     Organic or non-inorganic extraction removes
    proteins.
    II. DNA Fragmentation

     Cut the DNA into different sized pieces.


     Use restriction endonucleases (RE)
     Nucleases hydrolyze the bonds that connect
    bases within the strand, resulting in cleavage of
    the strand.
     They cleave the double stranded nucleic acid
    only at specific points In vivo, they are involved
    in DNA metabolism and repair or in bacterial host
    defense.
    III. Gel Electrophoresis

    Sorts the DNA pieces by size


     Gels are solid with microscopic pores
     Agarose or polyacrimide

     Gel is soaked in a buffer which controls

    the size of the pores


     Standards should also be run
    IV. Blotting

     Transfer the DNA from the gel to a solid


    support.
     The blot is usually done on a sheet of

    nitrocellulose paper or nylon.


     DNA is then neutralized with NaCl to prevent
    re-hybridization before adding the probe.
     Transferred by either electrophoresis or capillary
    blotting.
    Figure Southern blotting
    VII. Hybridization

     The labeled probe is added to the blocked


    membrane in buffer and incubated for
    several hours to allow the probe molecules
    to find their targets.
    VIII. Washing

     Excess probe will have bound nonspecifically to


    the membrane despite the blocking reagents.
     Blot is incubated with wash buffers containing
    NaCl and detergent to wash away excess probe
    and reduce background.
    IX. Detection

     Radioactive probes enable


    autoradiographic detection.
    Would you watch the animation?

    A. Yes
    B. No
    SUMMARY OF PROCEDURE

    1. Extract and purify DNA from cells


    2. DNA is restricted with enzymes
    3. Sort by electrophoresis
    4. Denature DNA
    5. Transfer to nitrocellulose paper
    6. Block with excess DNA
    7. Wash off unbound probe
    8. Autoradiograph

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