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  Section: Biotechnology Methods » Microbiology
 
 
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Extraction of Bacterial DNA

 
     
 
Content
Microbiology
  Introduction
  The Microscopy
  The Bright Field Microscope
  Introduction to the Microscope and Comparison of Sizes and Shapes of Microorganisms
  Cell Size Measurements: Ocular and Stage Micrometers
  Measuring Depth
  Measuring Area
  Cell Count by Hemocytometer or Measuring Volume
  Measurement of Cell Organelles
  Use of Darkfield Illumination
  The Phase Contrast Microscope
  The Inverted Phase Microscope
  Aseptic Technique and Transfer of Microorganisms
  Control of Microorganisms by using Physical Agents
  Control of Microorganisms by using Disinfectants and Antiseptics
  Control of Microorganisms by using Antimicrobial Chemotherapy
  Isolation of Pure Cultures from a Mixed Population
  Bacterial Staining
  Direct Stain and Indirect Stain
  Gram Stain and Capsule Stain
  Endospore Staining and Bacterial Motility
  Enumeration of Microorganisms
  Biochemical Test for Identification of Bacteria
  Triple Sugar Iron Test
  Starch Hydrolysis Test (II Method)
  Gelatin Hydrolysis Test
  Catalase Test
  Oxidase Test
  IMVIC Test
  Extraction of Bacterial DNA
  Medically Significant Gram–Positive Cocci (GPC)
  Protozoans, Fungi, and Animal Parasites
  The Fungi, Part 1–The Yeasts
  Performance Objectives
  The Fungi, Part 2—The Molds
  Viruses: The Bacteriophages
  Serology, Part 1–Direct Serologic Testing
  Serology, Part 2–Indirect Serologic Testing

Objectives
  • Describe the DNA within bacterial cells.
  • Perform a DNA extraction and isolate a DNA molecule.
Introduction
In this activity, you will extract a mass of DNA from bacterial cells visible to the naked eye.
  1. The preparation of DNA from any cell type, bacterial or human, involves the same general steps:
    1. Disrupting the cell (and nuclear membrane, if applicable),
    2. Removing proteins that entwine the DNA and other cell debris, and
    3. Doing a final purification.
      1. These steps can be accomplished in several different ways, but are much simpler than expected. The method chosen generally depends upon how pure the final DNA sample is and how accessible the DNA is within the cell.
      2. Bacterial DNA is protected only by the cell wall and cell membrane; there is no nuclear membrane as in eukaryotic cells. Therefore, the membrane can be disrupted by using dishwashing detergent, which dissolves the phospholipid membrane, just as detergent dissolves fats from a frying pan. (The process of breaking open a cell is called cell lysis.) As the cell membranes dissolve, the cell contents flow out, forming a soup of nucleic acid, dissolved membranes, cell proteins, and other cell contents, which is referred to as a cell lysate. Additional treatment is required for cells with walls, such as plant cells and bacterial cells that have thicker, more protective cell walls (such as Gram-positive or acid-fast organisms). Additional treatments may include enzymatic digestion of the cell wall or physical disruption by means such as blending, sonication, or grinding.

  2. After cell lysis, the next step involves purifying the DNA by removing proteins (histones) from the nucleic acid. Treatment with protein-digesting enzymes (proteinases) and/or extractions with the organic solvent phenol are 2 common methods of protein removal. Because proteins dissolve in the solvent but DNA does not, and because the solvent and water do not mix, the DNA can be physically separated from the solvent and proteins.
  3. In this activity, you will not attempt any DNA purification: your goal is simply to see the DNA. You will lyse E. coli with detergent and layer a small amount of alcohol on top of the cell lysate. Because DNA is insoluble in alcohol, it will form a white, web-like mass (precipitate) where the alcohol and water layers meet. Moving a glass rod up and down through the layers allows you to collect the precipitated DNA. But this DNA is very impure, mixed with cell debris and protein fibers.
  4. Before you begin the DNA isolation, make sure you know the procedure to follow. Draw out a flow chart below including the amount of each reagent and the time for that part of the procedure.
Materials


Methods

  1. Apply your PPE, including eye protection, for this lab. Locate the water baths and the ice-cold ethanol. Determine a method for timing the various steps.
  2. Label a 5-mL disposable tube and fill it with exactly 3 mL of distilled water. Using a swab, inoculate E. coli from the stock culture and agitate it in the 3 mL of distilled water.
  3. Add 3 mL of the detergent to the suspension of E.coli. Mix each tube by gently shaking.
  4. Place each tube into the water bath for 15 minutes.

    Maintain the water bath temperature above 60°C but below 70°C. A temperature higher than 60°C is needed to destroy the enzymes that degrade DNA.

  5. Cool the tube in an ice bath until it reaches room temperature.
  6. The next step involves precipitating the DNA by using solvent. Carefully pipete 3 mL of ice-cold ethanol (it may be in the freezer) on top of the detergent and E. coli suspension mixture. The alcohol should float on top and not mix. (It will mix if you stir it or squirt it in too fast, so be careful.) Water-soluble DNA is insoluble in alcohol and precipitates when it comes in contact with it.
  7. By carefully placing a clean glass rod through the alcohol into the suspension, a web-like mass will become evident; this mass is precipitated DNA. The rod carries a little alcohol into the suspension, precipitating and attaching to the DNA. Do not totally mix the 2 layers.

 
     
 
 
     




     
 
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