DNA Extraction Methods from Plants
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Questions and Answers

What is the first step in the DNA isolation process?

  • Resuspend DNA
  • Sample Collection (correct)
  • Cell Lysis
  • DNA Precipitation

    • Which buffer is commonly used to resuspend the DNA pellet for storage?

  • LB broth
  • Saline solution
  • TE buffer (correct)
  • PBS buffer

    • At what temperature should DNA be stored for short-term use?

  • -20°C
  • Room temperature
  • 4°C (correct)
  • -80°C

    • What is the purpose of washing the DNA with 70% ethanol?

    <p>To remove contaminants</p> Signup and view all the answers

    During the initial steps of DNA extraction from whole blood, what is the primary aim during RBC lysis?

    <p>To remove red blood cells</p> Signup and view all the answers

    What is the primary purpose of cell lysis in DNA extraction from plant cells?

    <p>To release DNA into the solution.</p> Signup and view all the answers

    Which of the following agents is commonly used to disrupt the cell membrane during DNA isolation?

    <p>SDS or Triton X-100</p> Signup and view all the answers

    What is the role of RNases in the DNA extraction process?

    <p>To digest RNAs that could interfere with DNA extraction.</p> Signup and view all the answers

    What is the final appearance of DNA after precipitation during the extraction process?

    <p>A white, stringy material.</p> Signup and view all the answers

    What is the purpose of washing the DNA pellet with 70% ethanol?

    <p>To remove residual salts and contaminants.</p> Signup and view all the answers

    What must be done to plant tissues that are particularly tough during DNA extraction?

    <p>Use homogenization in conjunction with lysis buffer.</p> Signup and view all the answers

    Why is the use of EDTA important in the DNA extraction process?

    <p>It inhibits DNases which could degrade DNA.</p> Signup and view all the answers

    What is the main goal of DNA extraction?

    <p>To study and analyze the isolated DNA.</p> Signup and view all the answers

    What is the primary purpose of ammonium chloride in RBCs lysis?

    <p>To provide a hypotonic solution that disrupts RBCs</p> Signup and view all the answers

    Which component is responsible for maintaining the pH of the RBCs lysis buffer?

    <p>Potassium bicarbonate</p> Signup and view all the answers

    What role does EDTA play in both RBCs and WBCs lysis?

    <p>Inhibiting DNases</p> Signup and view all the answers

    What is the function of using a detergent like SDS in WBCs lysis?

    <p>To disrupt the cell membrane</p> Signup and view all the answers

    After lysing cells, what is the subsequent step to remove proteins and contaminants from DNA?

    <p>Adding ammonium acetate</p> Signup and view all the answers

    What is the main purpose of adding cold ethanol or isopropanol in the DNA precipitation step?

    <p>To precipitate DNA from the supernatant</p> Signup and view all the answers

    What happens to the RBCs when they are treated with a hypotonic lysis buffer?

    <p>They swell and eventually burst</p> Signup and view all the answers

    Which process is used to separate the DNA after proteins have been precipitated?

    <p>Centrifugation</p> Signup and view all the answers

    What is the purpose of adding PCI to the lysate during the DNA extraction process?

    <p>To separate phases</p> Signup and view all the answers

    What is the order of the main phases formed after adding PCI to the lysate?

    <p>Organic Phase, Interface, Aqueous Phase</p> Signup and view all the answers

    Which step follows taking the aqueous phase to a new tube in the DNA extraction process?

    <p>Add ice-cold ethanol</p> Signup and view all the answers

    What is the primary aim of washing the DNA with 70% ethanol?

    <p>To remove residual salts and contaminants</p> Signup and view all the answers

    What does the 70% ethanol wash accomplish in the PCIA DNA extraction method?

    <p>Purify the DNA</p> Signup and view all the answers

    Which buffer is recommended for resuspending purified DNA?

    <p>TE buffer (Tris-EDTA)</p> Signup and view all the answers

    When extracting DNA from animal tissue, what is the first step to prepare the sample?

    <p>Add lysis buffer and grind the tissue</p> Signup and view all the answers

    What happens to the DNA after adding ice-cold ethanol or isopropanol?

    <p>It precipitates out of the solution</p> Signup and view all the answers

    Which step follows the DNA precipitation in the whole blood DNA isolation method?

    <p>Wash DNA</p> Signup and view all the answers

    What role does isoamyl alcohol play in the PCI method for DNA extraction?

    <p>It improves phase separation by reducing foaming</p> Signup and view all the answers

    What critical role does the centrifugation step serve in the DNA extraction process?

    <p>It separates the DNA from the cells</p> Signup and view all the answers

    What is the primary function of phenol in the PCI method?

    <p>To denature proteins and dissolve lipids</p> Signup and view all the answers

    After washing the precipitated DNA with ethanol, what is the final storage solution recommended?

    <p>TE buffer or water</p> Signup and view all the answers

    Which of the following is NOT a step in the whole blood DNA isolation method?

    <p>Cellular lipid extraction</p> Signup and view all the answers

    Why is adjusting the pH important during DNA extraction methods such as PCI?

    <p>To maintain DNA's structural integrity</p> Signup and view all the answers

    Study Notes

    DNA Extraction Methods

    •  DNA extraction isolates DNA from cells for study. It involves collecting cells, breaking them open, removing proteins and other materials, and isolating pure DNA.
    • Different methods exist for extracting DNA depending on the source material (e.g., plant cells, animal cells, whole blood, cultured animal cells and tissues).

    DNA Extraction from Plant Cells

    •  Introduction: DNA extraction is the process of isolating DNA from plant cells to study it.
    • Main Steps:
      • Sample Collection & Preparation: Obtain a plant sample, such as tomato or guava.
      • Cell Lysis: Break open cells to release DNA using a lysis buffer with detergent (e.g., SDS, Triton X-100) to disrupt the cell membrane. For tougher tissues, homogenization (using a mortar and pestle or homogenizer) is also used. EDTA inhibits DNases. Salts like NaCl also are used.
      • Removal of Proteins and Other Contaminants: Separate proteins and other cellular debris from DNA using proteases (e.g., Proteinase K) and RNases. (Optional).
      • Precipitation of DNA: Add cold ethanol or isopropanol to the solution to precipitate the DNA out of the solution, forming a pellet.
      • Washing the DNA: Wash the DNA pellet with 70% ethanol to remove residual salts and organic compounds.
      • Resuspension and Storage: Dissolve the purified DNA in a suitable buffer (e.g., TE buffer or sterile distilled water) for storage. Short-term storage is at 4°C, while long-term storage is at -20°C or -80°C.

    DNA Extraction from Whole Blood

    • Introduction: Whole blood contains all blood components (RBCs, WBCs, platelets) without separation.
    • Main Steps:
      • Sample Collection & Preparation: Collect a blood sample.
      • RBCs Lysis: Remove red blood cells using RBCs lysis buffer (containing ammonium chloride, potassium/sodium bicarbonate, and EDTA).
      • WBCs Lysis: Break open the remaining white blood cells using a lysis buffer containing detergent (such as SDS), EDTA and salt (NaCl).
      • Removal of Proteins and Other Contaminants: Separate proteins and other cellular debris using a method. For example, separating proteins using ammonium acetate,
      • DNA Precipitation: Precipitate DNA from supernatant using cold ethanol or isopropanol.
      • Washing the DNA: Wash the DNA pellet using 70% ethanol.
      • Resuspension and Storage: Resuspend the purified DNA in a suitable buffer (e.g., TE buffer or sterile distilled water). Store at 4°C for short-term or -20°C/-80°C for long-term.

    DNA Extraction from Cultured Animal Cells & Animal Tissues using Phenol:Chloroform:Isoamyl Alcohol (PCI) Method

    • Introduction: DNA extraction is commonly performed using PCI method due to its high yield.
    • Key Components and Roles:
      • Phenol (25): A hydrophobic and denser than water; separates proteins and lipids from DNA.
      • Chloroform (24): Improves efficiency and clarity of phase separation; helps to make proteins and lipids more soluble.
      • Isoamyl Alcohol (1): Reduces foaming during the mixing of phenol and chloroform.
    • Main Steps:
      • Sample Collection & Preparation: Collect the cells/tissue.
      • Cell Lysis & Homogenization: Break open cells and homogenize the tissue using lysis buffer.
      • PCI Addition: Add PCI to the cell lysate. This will create three phases: DNA (aqueous phase), proteins (interface), and lipids (organic phase).
      • Separation/Precipitation: Take the aqueous phase, then precipitate DNA using ice-cold ethanol or isopropanol.
      • Washing & Storage: Wash the DNA pellet to remove salts, and resuspend in a suitable buffer for storage.
    • Different protocols may be involved for different types of tissues.

    Additional Notes

    • Various methods and protocols are used in DNA extraction, each with advantages and disadvantages.
    • Proper handling and storage of DNA are important.
    • Several solutions/chemicals like EDTA (Ethylenediaminetetraacetic acid), TritonX-100, Proteinase K and RNase, are involved in the process.
    • This information covers common methods for various sample types used in molecular biology.

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    Description

    Explore the various methods of DNA extraction specifically from plant cells. This quiz covers the essential steps such as sample collection, cell lysis, and purification techniques. Understand the importance of different reagents and their roles in isolating pure DNA for further study.

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