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    Causes of Using Chemical Compound in DNA Isolation From Plant Sample

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    The document discusses steps in the DNA isolation process from plant samples using the CTAB method. It describes the roles of various chemicals used, including TE buffer to protect DNA, phenol and chloroform to remove proteins and lipids, isopropanol to precipitate DNA, ethanol as a dehydrating agent for precipitation, and sodium acetate to neutralize charges on DNA allowing precipitation. Multiple centrifugation steps are used to purify the DNA at various points in the process.

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    Causes of Using Chemical Compound in DNA Isolation From Plant Sample

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    pijushgen
    5K views3 pages
    The document discusses steps in the DNA isolation process from plant samples using the CTAB method. It describes the roles of various chemicals used, including TE buffer to protect DNA, phenol and chloroform to remove proteins and lipids, isopropanol to precipitate DNA, ethanol as a dehydrating agent for precipitation, and sodium acetate to neutralize charges on DNA allowing precipitation. Multiple centrifugation steps are used to purify the DNA at various points in the process.

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    Role of Using Chemical Compound in DNA Isolation From Plant Sample by CTAB Method

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    The document discusses steps in the DNA isolation process from plant samples using the CTAB method. It describes the roles of various chemicals used, including TE buffer to protect DNA, phenol and chloroform to remove proteins and lipids, isopropanol to precipitate DNA, ethanol as a dehydrating agent for precipitation, and sodium acetate to neutralize charges on DNA allowing precipitation. Multiple centrifugation steps are used to purify the DNA at various points in the process.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOC, PDF, TXT or read online from Scribd
    Download as doc, pdf, or txt
    5K views3 pages

    Causes of Using Chemical Compound in DNA Isolation From Plant Sample

    Uploaded by

    pijushgen
    The document discusses steps in the DNA isolation process from plant samples using the CTAB method. It describes the roles of various chemicals used, including TE buffer to protect DNA, phenol and chloroform to remove proteins and lipids, isopropanol to precipitate DNA, ethanol as a dehydrating agent for precipitation, and sodium acetate to neutralize charges on DNA allowing precipitation. Multiple centrifugation steps are used to purify the DNA at various points in the process.

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     DNA Isolation from plant sample by CTAB

    method
     Causes of using chemical compound in DNA
    isolation

    • What is “TE”stands for?


    Tris-EDTA (Ethylenediamine Tetraacetic Acid; buffered solution).
    "TE" is derived from its components: Tris, a common pH buffer, and
    EDTA, a molecule chelating cations like Mg2+. The purpose of TE buffer
    is to protect DNA or RNA from degradation.

    • What is the role of TE buffer in DNA isolation?


    The purpose of TE buffer is to protect DNA or RNA from degradation. It
    is a buffer for storage of DNA & RNA.

    Role of phenol,chloroform in DNA isolation?


    Phenol- phenol is used to denature the proteins. Or
    phenol helps to remove non polar proteins and lipids from the solution.
    chloroform - prevent shearing of dna during isolation. It solubilizes
    lipids and a lot of proteins to remove them from the DNA.

    Phenol -chloroform and isoamylalcohol role in dna


    isolation?
    The phenol, chloroform and also isoamyl alchohol are added in a specific ratio
    of 25: 24:1.
    Isoamylalchohol, (CH3)2CHCH2CH2OH or Isopentanol :Used to prevent
    phosgene(COCl2, Carbonyl chloride) from reaction of chloroform(CHCl3) with
    air.

    The Phenol:Chloroform:Isoamylalchohol (PCI) solution is added to the cell


    extract after removal removal of debris. After proper mixing, cetrifugation is
    done to separate the phases. Two phases are formed: The upper, the
    aqueous phase that contains DNA, the lower phase, that phenol phase, that
    contains organic impurities. Thus two phases are separated by a very clearly
    defined boundary of coagulated proteins.
    The aqueous phase is precipitated and then the DNA could be pelleted after
    rounds of purifications.

    What is the role of Isopropanol in DNA isolation?


    Its main role is to pricipitate the DNA, by engaging with the water molecule
    as not giving chance for the DNA to get dissolve in the water.

    • What is the role of ethanole in dna isolation?


    Ethanol is a dehydrating agent. In DNA isolation it is used for the precipitation
    of DNA molecule. When a molecule is to be precipitated it should be
    dehydrated as the water molecules forming a film around it prevent their
    interaction. In DNA isolation the DNA molecules are dehydrated by ethanol.

    • Why Discard the supernatant in early stage of


    dna isolation?
    Pieces of cell wall are released from the bacteria and are floating around in the
    supernatant. These cell wall pieces can inhibit enzyme action on your final DNA, so it
    is important to get rid of all of the supernatant and to even invert the tube.

    • What is the role of sodium acetate in DNA


    extraction?
    The function of adding the acetate solution (either sodium acetate or
    ammonium acetate) is to raise the salt concentration, and therefore cause
    the nucleic acid (here DNA) to precipitate out of solution.
    The role of the salt in the protocol is to neutralize the charges on the sugar
    phosphate backbone. A commonly used salt is sodium acetate. In solution,
    sodium acetate breaks up into Na+ and [CH3COO]-. The positively charged
    sodium ions neutralize the negative charge on the PO3- groups on the nucleic
    acids, making the molecule far less hydrophilic, and therefore much less
    soluble in water.

    DNA extraction - why 100% and 70% ethanol is


    used?

    I added 100% ethanol I have to "incubate" the samples in the freezer (-20) for
    about a half an hour, but then I have to to the 70% step, and here I only have
    to centrifuge the samples, not "incubate".
    Is the 75% step simple an extra purification step to get rid of some 100%
    ethanol and other stuff.

    Or says to,

    70% ethanol to remove salts and small organic molecules, and


    resuspended in buffer at a concentration suitable for further
    experimentation.

    • Why so many centrifuge is used?


    Cause to get purify DNA.
     BY PIJUSH SUTRADHAR

    Genetic engineering &


    Biotechnology Dept.

    3rd year 1st semester

    • REG.2007432026

     Email: [email protected]

    [email protected]

     Contact no: 01719232566

    SHAHJALAL UNIVERSITY OF SCIENCE AND TECHNOLOGY

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