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    Home > Biochemistry News > Biotechnology News > The horizontal agarose gel electrophoresis method detects DNA

    The horizontal agarose gel electrophoresis method detects DNA

    • Last Update: 2020-11-03
    • Source: Internet
    • Author: User
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    related topics
    the purpose ofLearn the purity
    configuration
    content, and molecular weight of
    DNA
    in horizontal
    agar gel electrophoresis.Experimental PrinciplesClosed ring-shaped, mitochondrial and open-ringed granulate DNA, due to their different configuration, show different migration rates on agarose
    gel electrophoresis
    of abrominated ethyl ingots, so that observations under ultraviolet light can distinguish between closed ring-like proton DNA (cccDNA), mitochondrial DNA (L-DNA) and open-loop granulated DNA (ocDNA).test materials
    reagents
    (i) experimental samples seeds
    pUC118
    (ii) reagents
    1. l DNA/HindIII Molecular Weight Standard
    2. Bromphenol blue indicator dot buffer
    0.2% bromophenol blue
    50% sucrose
    3. 1mg/ml ethyl bromide ingot solution
    4. Electrophoretic buffer (TAE)
    40 mmol/L Tris-acetic acid
    1 mol/L
    EDTA
    (preparation method: 24.2 g Tris alkali, 5.71 ml ice acetic acid, 10 ml 0.5mol/L EDTA (pH8.0), fixed to 5000ml)
    5. 0.7% agarose gel
    (preparation method: 0.35 g of agarose, add 50 ml TAE electrophoresis buffer)
    (iii) instrument
    trace
    -capacitor
    electrophoresis instrument
    horizontal electrophoresis tank transmission ultraviolet observerexperimental step
    1. Select the right horizontal electrophoresis tank and adjust the electrophoresis slot plane to the level. Check the line between the regulator power supply and the positive and negative poles.
    2. Select the right aperture size dot comb, vertically mounted at one end of the electrophoresis mold, so that the bottom of the dot comb from the bottom of the electrophoresis mold distance of 1.0mm.
    3. Prepare 0.7% agarose gel, 100 degrees C water bath
    heat
    until agarose melts evenly.
    4. Using
    straw
    take a small amount of agarose gel solution to seal the electrophoresis mold around, to prevent penetration when watering agarose gel plate. When the agarose gel is cooled to about 60 degrees C, add a drop of ethyl bromide, shake well and gently pour into the electrophoresis mold, the thickness of the agarose gel is 3 to 5mm. When pouring glue to avoid the production of bubbles, if there are bubbles can be carefully sucked out of the straw.
    5. After the agarose gel solidifies, place at room temperature for 20 minutes, carefully unplug the bezels at both ends of the sample comb and electrophoresis mold, and keep the sample holes intact.
    6. Put the electrophoresis mold into the electrophoresis tank and add the electrophoresis buffer so that the electrophoresis buffer surface is 1 to 2 mm above the surface of the agarose gel. If there are bubbles in the sample hole, use the straw carefully sucked out, so as not to affect the addition.
    7. Mix 15 ml of DNA samples with a 1/5 volume bromophenol blue indicator sample buffer. The sample buffer can not only improve the density of the sample, so that the sample evenly sank into the sample hole, but also make the sample color, easy to sample and estimate the electrophoresis time and determine the location of electrophoresis.
    8. Carefully add the sample to the sample addition hole with a trace shifter to record the sample order.
    9. Cover the electrophoresis tank, turn on the power switch, the maximum voltage does not exceed 5V/cm (100 to 150V constant electrophoresis), so that the DNA from the negative to the positive.
    10. The electrophoresis time varies according to the specific requirements of the experiment. Electrophoresis usually takes 1 to 3 hours. Turn off the power after electrophoresis, wear disposable plastic
    gloves
    remove the gel, as far as possible, all electrophoresis buffer dried, in 254nm wavelength transmission UV lamp observation.(
    Tip
    )
    (i) agarose gel electrophoresis
    1. Nature of agarose gel
    Agarose is a straight-chain polysaccharose extracted from seaweed, which consists of D-semi-lactose and 3,6-dehydrated-L-semi-lactose residues alternately arranged into a line polysaccharose. When agar is heated to about 90 to 100 degrees C, a clear and transparent liquid can be formed. When poured over the template and cooled to 40 to 45 degrees C, the gel is solidified. Agarose is hydromassive, does not contain charged clumps, does not cause DNA degeneration, and does not adsorption of isolated substances, so it is a good gel. Agarose gels distinguish between DNA fragments with a difference of 100bp.
    2. The migration rate of DNA molecules
    The migration rate of DNA molecules in electrophoresis is multi-factored, in addition to determining the size and configuration of DNA molecules, but also the concentration of agarose gel, voltage size, buffer pH and temperature during electrophoresis.
    (2) The problems paid attention to in the experimental operation
    1. When heating dissolved agar sugar, the container should be constantly shaken so that the particles attached to the wall are also completely dissolved.
    2. Ethyl bromide is a strong carcinogen and moderately toxic, so care must be taken. Be sure to wear gloves when operating, used gloves to timely turn the gloves over hand, so that the pollution of ethyl bromide ingot face in.
    3. UV light at 254nm wavelengths was clearer than 366nm, but the amount of indip DNA produced was also higher. Ultraviolet light is harmful to the eyes and should be observed with glasses or a protective mask.
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