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    Mipa - PB Slide Review On Cloning Technique

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    PriawanIndra
    This document summarizes the basic steps for cloning genes: 1. Isolation of DNA from organisms through techniques like gel electrophoresis to separate DNA fragments by size 2. Characterization of the isolated DNA using methods like Southern blotting 3. Insertion of the DNA fragments into vectors through processes like ligation or transformation for propagation in host cells

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    Mipa - PB Slide Review On Cloning Technique

    Uploaded by

    PriawanIndra
    23 views36 pages
    This document summarizes the basic steps for cloning genes: 1. Isolation of DNA from organisms through techniques like gel electrophoresis to separate DNA fragments by size 2. Characterization of the isolated DNA using methods like Southern blotting 3. Insertion of the DNA fragments into vectors through processes like ligation or transformation for propagation in host cells

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    mipa_-_pb_slide_review_on_cloning_technique

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    This document summarizes the basic steps for cloning genes: 1. Isolation of DNA from organisms through techniques like gel electrophoresis to separate DNA fragments by size 2. Characterization of the isolated DNA using methods like Southern blotting 3. Insertion of the DNA fragments into vectors through processes like ligation or transformation for propagation in host cells

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    23 views36 pages

    Mipa - PB Slide Review On Cloning Technique

    Uploaded by

    PriawanIndra
    This document summarizes the basic steps for cloning genes: 1. Isolation of DNA from organisms through techniques like gel electrophoresis to separate DNA fragments by size 2. Characterization of the isolated DNA using methods like Southern blotting 3. Insertion of the DNA fragments into vectors through processes like ligation or transformation for propagation in host cells

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 36

    Review on cloning technique

    Lecture 9
    How to clone genes
    1. Isolation DNA
    2. Characterization
    3. Insertion into vector
    4. Transformation
    5. Expression / Selection
    1. Isolation of DNA
    2. Characterization
    • Southern Blot
    • DNA is negatively charged.
    • When placed in an electrical field, DNA will
    migrate toward the positive pole (anode).
    • An agarose gel is used to slow the movement
    of DNA and separate by size
    • Polymerized agarose is porous,
    allowing for the movement of DNA
    Scanning Electron Micrograph of
    Agarose Gel (1×1 µm) 
    Electrophoresis

    DNA

    - ++

    Power
    How fast will the DNA migrate?
    • strength of the electrical field, buffer,
    density of agarose gel…
    • Size of the DNA!
    • *Small DNA move faster than large DNA
    • …gel electrophoresis separates DNA according
    to size
    Electrophoresis, contd
    DNA

    small
    large
    - +

    Power
    Agarose

    3,6-anhydro
    D-galactose L-galactose
    •Sweetened agarose gels
    have been eaten in the Far
    East since the 17th century.

    •Agarose was first used in


    Agarose is a linear polymer
    biology when Robert Koch*
    extracted from seaweed.
    used it as a culture medium
    for Tuberculosis bacteria in
    1882
    Making an Agarose Gel

    An agarose gel is prepared by


    combining agarose powder
    and a buffer solution.

    Flask for boiling


    Electrophoresis Equipment

    
    Power supply

    Cover
    
    Gel tank
    Electrical leads
    

    
    Casting tray
    Gel combs
    Gel casting tray & combs
    Preparing the Casting Tray

    Seal the edges of the casting tray and put in the combs.
    Place the casting tray on a level surface. None of the gel
    combs should be touching the surface of the casting tray.
    Agarose Buffer Solution

    Combine the agarose powder and buffer solution.


    Use a flask that is several times larger than the
    volume of buffer.
    Melting the Agarose

    Agarose is insoluble at The agarose solution is boiled


    room temperature until clear
    Gently swirl the solution periodically when heating to allow all
    the grains of agarose to dissolve.
    ***Be careful when boiling - the agarose solution may become
    superheated and may boil violently if it has been heated too
    long in a microwave oven.
    Pouring the gel

    Allow the agarose solution to cool slightly (~60ºC) and


    then carefully pour the melted agarose solution into the
    casting tray. Avoid air bubbles.
    When cooled, the agarose polymerizes, forming a flexible gel.
    It should appear lighter in color when completely cooled (30-
    45 minutes). Carefully remove the combs and tape.
    Place the gel in the electrophoresis chamber
    A HORIZONTAL ELECTROPHORESIS
    Vertical slab gel apparatus
    DNA

    buffer     
    wells

    Cathode Anode 
    (negative) (positive)

    Add enough electrophoresis buffer to cover the gel to a


    depth of at least 1 mm. Make sure each well is filled with
    buffer.
    Sample Preparation
    • Mix the samples of DNA with the 6X sample
    loading buffer (w/ tracking dye). This allows
    the samples to be seen when loading onto the
    gel, and increases the density of the samples,
    causing them to sink into the gel wells.

    6X Loading Buffer: 
    • Bromophenol Blue (for color)
    • Glycerol (for weight)
    Loading the Gel

    Carefully place the pipette tip over a well and gently expel
    the sample. The sample should sink into the well. Be
    careful not to puncture the gel with the pipette tip.
    Running the Gel

    Place the cover on the electrophoresis chamber, connecting the


    electrical leads. Connect the electrical leads to the power
    supply. Be sure the leads are attached correctly - DNA migrates
    toward the anode (red). When the power is turned on, bubbles
    should form on the electrodes in the electrophoresis chamber.
    Cathode
    (-)

     wells
    DNA  Bromophenol Blue
    (-)
    

    Anode Gel
    (+)

    After the current is applied, make sure the Gel is running


    in the correct direction. Bromophenol blue will run in the
    same direction as the DNA.
    Inclusion of a DNA ladder (DNAs of know sizes) on the gel
    makes it easy to determine the sizes of unknown DNAs.
    Staining the Gel
    Ethidium bromide binds to DNA and fluoresces under UV light,
    allowing the visualization of DNA on a Gel.
    • Ethidium bromide can be added to the gel and/or running
    buffer before the gel is run or the gel can be stained after it has
    run.

    ***CAUTION! Ethidium bromide


    is a powerful mutagen and is
    moderately toxic. Gloves
    should be worn at all times.
    Safer alternatives to Ethidium Bromide

    • Methylene Blue
    • BioRAD - Bio-Safe DNA Stain
    • Ward’s - QUIKView DNA Stain
    • Carolina BLU Stain

    advantages
    Inexpensive disadvantages
    Less toxic Less sensitive
    No UV light required More DNA needed on gel
    No hazardous waste Longer staining/destaining time
    disposal
    Staining the Gel

    Place the gel in the staining tray containing warm diluted stain.
    • Allow the gel to stain for 25-30 minutes.
    • To remove excess stain, allow the gel to destain in water.
    • Replace water several times for efficient destain.
    Ethidium Bromide requires an ultraviolet light source to
    visualize
    DNA ladder DNA ladder
     1 2 3 4 5 6 7 8 
    wells
    wells

     5,000 bp
     2,000
     1,650
     1,000
     850
     650
     500
    PCRPCR
    Product
    Product
     300
     200
     100

    + - - + - + + -
    Southern Blotting
    If a specific fragment of DNA is desired, it can be cut out as
    a lump of gel and removed from the gel by diffusion into a small
    volume of water.
    insertion into prokaryotic or eukaryotic cells
    4. Transformation
    5. Expression/ Selection
    • Based on marker of vector
    - antibiotic resistence
    - expression of gene ie. X-gal gene
    - etc ???
    Scale up / Production
    • Fermentation
    • Other cultivation

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