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    DNA Isolation

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    romaehab201912
    Plasmid DNA was isolated from bacterial cells using alkaline lysis. The procedure involved lysing the cells with SDS and NaOH solutions to release the plasmid DNA, followed by precipitation of the DNA with potassium acetate and isopropanol. The isolated plasmid DNA was analyzed by agarose gel electrophoresis, which showed two bands corresponding to supercoiled and relaxed plasmid forms, as well as a band for RNA. Proper isolation of plasmid DNA is important for downstream applications like PCR and DNA sequencing.

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    DNA Isolation

    Uploaded by

    romaehab201912
    6 views10 pages
    Plasmid DNA was isolated from bacterial cells using alkaline lysis. The procedure involved lysing the cells with SDS and NaOH solutions to release the plasmid DNA, followed by precipitation of the DNA with potassium acetate and isopropanol. The isolated plasmid DNA was analyzed by agarose gel electrophoresis, which showed two bands corresponding to supercoiled and relaxed plasmid forms, as well as a band for RNA. Proper isolation of plasmid DNA is important for downstream applications like PCR and DNA sequencing.

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    DNA_Isolation[1]

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    Plasmid DNA was isolated from bacterial cells using alkaline lysis. The procedure involved lysing the cells with SDS and NaOH solutions to release the plasmid DNA, followed by precipitation of the DNA with potassium acetate and isopropanol. The isolated plasmid DNA was analyzed by agarose gel electrophoresis, which showed two bands corresponding to supercoiled and relaxed plasmid forms, as well as a band for RNA. Proper isolation of plasmid DNA is important for downstream applications like PCR and DNA sequencing.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    6 views10 pages

    DNA Isolation

    Uploaded by

    romaehab201912
    Plasmid DNA was isolated from bacterial cells using alkaline lysis. The procedure involved lysing the cells with SDS and NaOH solutions to release the plasmid DNA, followed by precipitation of the DNA with potassium acetate and isopropanol. The isolated plasmid DNA was analyzed by agarose gel electrophoresis, which showed two bands corresponding to supercoiled and relaxed plasmid forms, as well as a band for RNA. Proper isolation of plasmid DNA is important for downstream applications like PCR and DNA sequencing.

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    © All Rights Reserved
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    Download as pptx, pdf, or txt
    of 10

    Isolation

    of DAN
    DNA isolation is a fundamental technique in
    molecular biology that allows scientists to obtain
    pure DNA samples for various applications. There
    are several methods available for DNA isolation,
    each with its advantages and limitations. The
    choice of method depends on the sample type, the
    intended application, and the resources available.
    Proper DNA isolation is crucial for accurate and
    reliable downstream analysis, such as PCR, DNA
    sequencing, and genetic engineering.
    Plasmids are small circular DNA molecules,
    most commonly found in bacterial cells (also
    found in Cyanobacteria and Fungi). They
    replicate in the cells independently of the
    chromosomes. The plasmids normally contain
    about 2% of total genetic information and
    multiply independently of the chromosome.
    Requirements for Isolation of Plasmid
    DNA
    • Tris, Sodium Dodecyl Sulphate (SDS),
    tryptone, yeast extract, sodium chloride
    (NaCl), agar, potassium- acetate
    • Boric acid, glucose, EDTA, sodium
    hydroxide.
    • Isopropanol, distilled water, high-speed
    refrigerated centrifuge, pH meter
    micropipettes, watch batch, vortex mixer
    • Agarose gel electrophoresis apparatus,
    Eppendorf tubes, Tips, glassware
    alkaline lysis plasmid isolation
    Preparation of Reagents
    The following solutions should be prepared in advance:
    • Solution I: Add 5 ml of 1M glucose (50mM final concentration of glucose), 0.25
    ml of 1M Tris (pH 8.0) (25 mM final concentration of Tris, pH 8.0) and 2 ml of 0.5
    M EDTA (pH 8.0) (10mM final concentration of EDTA). By using water make up
    the volume to 100 ml (autoclave the solution and store it at room temperature).
    • Solution II: Prepare a fresh solution on the day of the experiment by mixing 4 ml
    of 5N NaOH (0.2N final concentration of NaOH) and 10 ml of 10% SDS (final
    concentration of 1% SDS). By using autoclaved water make the volume of
    solution to 100 ml (discard the solution after use).
    • Solution III: Prepare a 3M potassium acetate solution of pH 4.8 (autoclave it and
    store it at 4°C).
    • Running Buffer (for electrophoresis): There are two common types of running
    buffers used in agarose gel electrophoresis: Tris-Borate-EDTA (TBE) and Tris-
    Acetate-EDTA (TAE).
    Procedure of Isolation of Plasmid DNA
    • After 24 hours of incubation, take 1.5 ml of culture from the 2 ml culture using an Eppendorf tube
    pipette.
    • Centrifuge the cells at 6000 rpm for 5-10 minutes. Discard the supernatant completely by inverting
    the Eppendorf tube on the blotting paper. Put the Eppendorf tube on ice.
    • Completely re-suspend the pellet in (0.1 ml) of ice-cold Solution I to get a uniform suspension. Put
    on ice for 5 minutes then keep at room temperature.
    • To this suspension, add 0.2 ml of freshly-prepared Solution II. The tube should be closed tightly.
    Properly mix the contents by inverting the tube five times.
    • Add 0.15 ml of ice-cold Solution III. close the tube tightly and mix the contents properly by
    inverting the tube. Keep for 5-7 minutes on ice. Vortex at 10000 to 12000 rpm for 10 minutes at 4°C
    • Soon transfer the supernatant to a fresh Eppendorf tube and add 0.45 ml of isopropanol. Gently mix
    by inverting the tube and keeping it at room temperature. Take out the supernatant.
    • Thereafter, add 0.1 ml of 70% ethanol into the pellet and spin at 10000 rpm for 5 minutes at 4°C.
    Then discard the supernatant carefully and dry the tube at 37°C. So that any traces of isopropanol
    could be removed.
    • Now, add 20 μl of IX-TE from the side and gently tap the tube with your fingers. Add 3.33 μl of 6X
    gel loading buffer and run on 1% agarose gel as it has been described below (Note: alternatively, a
    plasmid-isolation kit of any company can be used).
    Result of Isolation of Plasmid DNA
    After precipitating with isopropanol and congratulation, a white precipitate is
    observed on the sides or at the bottom of the centrifuge tube. Usually, 2
    bands are observed when the plasmid DNA is run on an agarose gel.
    These are the supercoiled and relaxed or open circular forms of the
    plasmid.
    During plasmid isolation no RNase was added, hence a band of RNA will
    also be seen on the gel. RNA moves faster than the DNA being small in
    size. Therefore, RNA can be distinguished from plasmid DNA.
    Video
    https://www.youtube.com/
    watch?v=SR4FX6O2u98
    Made by:
    Reem
    Mamdouh
    Romysaa Ehab

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