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    Agarose Gel Electrophoresis

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    Rian Lorenzo Mascarinas
    Agarose gel electrophoresis is used to separate DNA fragments by size. [1] A gel is made by dissolving agarose in TBE buffer and cooling it to solidify with wells for samples. [2] Ethidium bromide is added to stain the DNA fragments. [3] Samples mixed with dye are loaded and a current is applied to separate fragments by size, which are then viewed under UV light.

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    Agarose Gel Electrophoresis

    Uploaded by

    Rian Lorenzo Mascarinas
    66 views2 pages
    Agarose gel electrophoresis is used to separate DNA fragments by size. [1] A gel is made by dissolving agarose in TBE buffer and cooling it to solidify with wells for samples. [2] Ethidium bromide is added to stain the DNA fragments. [3] Samples mixed with dye are loaded and a current is applied to separate fragments by size, which are then viewed under UV light.

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    Agarose gel electrophoresis is used to separate DNA fragments by size. [1] A gel is made by dissolving agarose in TBE buffer and cooling it to solidify with wells for samples. [2] Ethidium bromide is added to stain the DNA fragments. [3] Samples mixed with dye are loaded and a current is applied to separate fragments by size, which are then viewed under UV light.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    66 views2 pages

    Agarose Gel Electrophoresis

    Uploaded by

    Rian Lorenzo Mascarinas
    Agarose gel electrophoresis is used to separate DNA fragments by size. [1] A gel is made by dissolving agarose in TBE buffer and cooling it to solidify with wells for samples. [2] Ethidium bromide is added to stain the DNA fragments. [3] Samples mixed with dye are loaded and a current is applied to separate fragments by size, which are then viewed under UV light.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 2

    AGAROSE GEL ELECTROPHORESIS

    (For the CSS451)

    Gel preparation

    1. Make a 1% agarose solution in 100ml 1 X TBE. A solution of up to 2-4% can be


    used if you analyze small DNA molecules, and for large molecules, a solution as
    low as 0.7% can be used.
    2. Carefully bring the solution just to the boil to dissolve the agarose in a microwave
    oven.
    3. Let the solution cool down to about 60 °C at room temperature, or water bath. Stir
    or swirl the solution while cooling.

    Wear gloves from here on, ethidium bromide is a mutagen, for more information on
    safety see ethidium bromide

    1. Add 5 µl ethidium bromide stock (10 mg/ml) per 100 ml gel solution for a final
    concentration of 0.5 ug/ml. Be very careful when handling the concentrated stock.
    Some researchers prefer not to add ethidium bromide to the gel itself, instead
    soaking the gel in an ethidium bromide solution after running.
    2. Stir the solution to disperse the ethidium bromide, then pour it into the gel rack.
    3. Insert the comb at one side of the gel, about 5-10 mm from the end of the gel.
    4. When the gel has cooled down and become solid, put the gel, together with the
    rack, into a tank with TAE. The gel must be completely covered with TAE, with
    the wells at the end electrode that will have the negative current.
    5. Carefully remove the comb. The holes that remain in the gel are the wells.

    Loading the gel:

    1. Injection of DNA ladder (molecular weight markers) into the first or the last well.
    2. Injection of samples into the other wells.

    For week 4 For Week 5


    2 ul DNA 20 ul PCR product
    15 ul H2O -
    3 ul 6X bromophenol blue 4 ul 6X bromophenol blue
    Total 20 ul Total 24 ul

    3. Close the lid of the electrophoresis chamber and apply current (100 V for 1hr
    minutes with 100 ml of gel). The DNA moves toward the positive anode due to
    the negative charges on its phosphate backbone.
    4. Check the gel under a UV light.

    1
    Note:

    The colored dye in the DNA ladder and DNA samples acts as a "front wave" that runs
    faster than the DNA itself. When the "front wave" approaches the end of the gel, the
    current is stopped. The DNA is stained with ethidium bromide, and is then visible under
    ultraviolet light.

    Small DNA strands move fast, large DNA strands move slowly through the gel. The
    DNA is not normally visible during this process, so the marker dye is added to the DNA
    to avoid the DNA being run entirely off the gel. The marker dye has a low molecular
    weight, and migrates faster than the DNA, so as long as the marker has not run past the
    end of the gel, the DNA will still be in the gel.

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