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    Dna Gel Electrophoresis: Gateway To Various Biotechnology Investigations

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    Nasrullah Roslan
    Gel electrophoresis is a technique used to separate DNA fragments by size. DNA has a negative charge and will migrate toward the positive electrode when placed in an agarose gel and an electric current is applied. Smaller fragments travel farther than larger ones. Ethidium bromide dye is often used to visualize the separated DNA bands under UV light. A DNA ladder containing fragments of known sizes allows estimation of unknown sample fragment lengths. Care must be taken in loading samples and running the electrophoresis for accurate results.

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    Dna Gel Electrophoresis: Gateway To Various Biotechnology Investigations

    Uploaded by

    Nasrullah Roslan
    92 views16 pages
    Gel electrophoresis is a technique used to separate DNA fragments by size. DNA has a negative charge and will migrate toward the positive electrode when placed in an agarose gel and an electric current is applied. Smaller fragments travel farther than larger ones. Ethidium bromide dye is often used to visualize the separated DNA bands under UV light. A DNA ladder containing fragments of known sizes allows estimation of unknown sample fragment lengths. Care must be taken in loading samples and running the electrophoresis for accurate results.

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    Gel Electrophoresis

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    Gel electrophoresis is a technique used to separate DNA fragments by size. DNA has a negative charge and will migrate toward the positive electrode when placed in an agarose gel and an electric current is applied. Smaller fragments travel farther than larger ones. Ethidium bromide dye is often used to visualize the separated DNA bands under UV light. A DNA ladder containing fragments of known sizes allows estimation of unknown sample fragment lengths. Care must be taken in loading samples and running the electrophoresis for accurate results.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    92 views16 pages

    Dna Gel Electrophoresis: Gateway To Various Biotechnology Investigations

    Uploaded by

    Nasrullah Roslan
    Gel electrophoresis is a technique used to separate DNA fragments by size. DNA has a negative charge and will migrate toward the positive electrode when placed in an agarose gel and an electric current is applied. Smaller fragments travel farther than larger ones. Ethidium bromide dye is often used to visualize the separated DNA bands under UV light. A DNA ladder containing fragments of known sizes allows estimation of unknown sample fragment lengths. Care must be taken in loading samples and running the electrophoresis for accurate results.

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    DNA GEL

    ELECTROPHORESIS
    Gateway to various biotechnology
    investigations
    What is Gel
    Electrophoresis?
    Electro- = flow of electricity,
    -phoresis = to carry across
    A gel is a colloid, a suspension of tiny
    particles in a medium, occurring in a solid
    form (like gelatin)
    Gel electrophoresis refers to the
    separation of charged particles located in a
    gel when an electric current is applied
    Charged particles can include DNA, amino
    acids, polypeptides, etc
    Why do gel electrophoresis?
    When DNA is cut by restriction enzymes*,
    the result is a mix of pieces of DNA of
    different lengths
    It is useful to be able to separate the pieces
    (for recovering particular pieces of DNA,
    for forensic work or for sequencing)

    *Reminder: Restriction enzymes are
    enzymes that cut up DNA.
    How does it work?
    DNA is an organic acid, and is negatively
    charged
    When the DNA is exposed to an electrical
    field, the particles migrate toward the
    positive electrode
    Smaller pieces of DNA can travel further in a
    given time than larger pieces
    What is needed?
    Agarose is a polysaccharide made
    from seaweed.
    It is dissolved in buffer and heated,
    then cools to a gelatinous solid with a
    network of crosslinked molecules
    Some gels are made with
    acrylamide if sharper bands are
    required (protein separation)
    Different gels
    Most agarose gels are made between 0.8% and
    2%.
    A 0.8% gel will show good resolution
    (separation) of large DNA fragments (5-
    10kb)
    A 2% gel will show good resolution for
    small fragments (0.2-1kb)
    Low percentage gels are very weak and may
    break when you try to lift them.
    High percentage gels are often brittle and do
    not set evenly.
    Buffer - either TBE or TAE
    The buffer provides ions in solution to
    ensure electrical conductivity
    Not only is the agarose dissolved in
    buffer, but the gel slab is submerged
    (submarine gel) in buffer after
    solidifying
    What is needed?
    Also needed
    are a power
    supply and a
    gel chamber
    What is needed?
    Gel electrophoresis
    Agarose gel
    - electrode + electrode
    DNA fragments
    buffer
    ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
    ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
    Gel electrophoresis
    - electrode + electrode
    current
    buffer
    ~~~~~~~~~~~~~~~~~~~~~~~~
    ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
    ~~~~~~~~~~~~~~~~~~~~~~~~
    Visualizing DNA
    Most of the time Ethidium bromide (EtBr) is
    used
    A fluorescent dye visualized when excited
    by UV light
    Intercalates into the DNA molecule,
    thereby staining it
    Gel is soaked in a solution of EtBr and the
    DNA bands take up the dye
    Then the gel is placed under UV light and
    visualized and/or photographed
    EtBr Gel Under UV Light
    **Each band that you see is a collection of millions of
    DNA molecules, all of the same length!!
    However
    Ethidium Bromide is a mutagen
    So we will use EtBr staining cards,
    which only carry trace amounts and
    minimize student exposure to the
    compound
    DNA ladders
    Frequently, one of
    the wells in your gel
    will contain a DNA
    ladder
    This is used as a
    marker to compare
    the sample DNA
    fragments and
    estimate their sizes
    Helpful Hints
    When placing the gel in the electrophoresis
    chamber:
    Make sure that the wells are closest to the
    negative (black) electrode (since DNA is
    negative)
    When adding the buffer to the chamber:
    Gently flood the gel from the end opposite the
    wells to minimize sample diffusion
    Before loading the wells:
    Orient the entire chamber close to the power
    supply so it is in place when the samples are
    ready to run
    Helpful Hints
    When loading samples in the wells of the gel:
    Use proper micropipette techniques
    Make a written record of which sample you
    will load in each well of the gel
    Be careful not to puncture the bottoms of the
    wells as you load the samples
    If you make a mistake, do NOT take more than
    the allotted sample; there are limited amounts

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