Chapter 14. Extracting, Isolating, and Visualizing DNA
Equipment and Materials
You’ll need the following items to complete this lab session. (The standard kit for this book, available from www.thehomescientist.com, includes the items listed in the first group.)
Materials from Kit
Goggles
Centrifuge tubes, 50 mL
Coverslips
Eosin Y stain
Funnel
Graduated cylinder, 10 mL
Methylene blue stain
Microscope slides (flat)
Pipettes
Reaction plate, 96-well
Scalpel
Sodium dodecyl sulfate, 10%
Spatula
Stirring rod
Test tubes
Test tube rack
Yeast (optional)
Materials You Provide
Gloves
Balance (optional)
Beef or pork liver, raw (or yeast)
Cheesecloth (or muslin, etc.)
Isopropanol (see text)
Freezer
Microscope
Paper towels
Saucer
Table salt
Teaspoon
Toothpick
Water, distilled
Background
All organisms are made up of cells, from the tiniest species such as single-cell bacteria and protozoa to the largest animals and plants, which are made up of trillions of individual cells. Every cell contains DNA, which is the hereditary genetic material that allows cells and organisms to function and to reproduce themselves.
With very few exceptions (research chimera on the Internet; the genetic kind, not the mythological kind) DNA taken from any cell of a particular individual of any species is identical to that taken from any other cell from that individual, and is unique to that individual. DNA found in unrelated members of the same species—for example, you and your best friend (assuming your best friend isn’t a dog or a diamond...) or two rosebushes—is nearly (but not quite) identical. DNA found in different but closely related species—for example, lions and tigers or wolves and coyotes—has greater differences, but is still extremely similar. DNA found in different, unrelated species—for example, a human and a cucumber—has still greater differences, but remains closely similar.
But what does DNA actually look like? Let’s find out. In this lab session, we’ll extract liver-cell DNA into a solution, isolate that DNA, and stain it to visualize it.
Procedure III-5-1: Extracting and Visualizing DNA
If you have not already done so, put on your goggles, gloves, and protective clothing.
Transfer five drops of methylene blue stain and five drops of eosin Y stain to one well of the reaction plate. Use a toothpick to mix the solutions thoroughly, and place the reaction plate aside for later use.
Make up 50 mL of normal saline by dissolving 0.45 g (one slightly rounded spatula spoon) of table salt in 50 mL of distilled water in a 50 mL centrifuge tube, and place the tube aside for later use.
Transfer about 40 mL of isopropanol to a second 50 mL centrifuge tube. Cap the tube and place it in the freezer to cool.
Obtain a teaspoon-size specimen of fresh beef or pork liver.
Note
It’s best to use fresh liver from a butcher or fresh market. Supermarket liver can be used but may yield inferior results, depending on its age. Also, you may substitute dried baker’s or brewer’s yeast for the liver, although lysis—breaking down the cell membranes and releasing the contents of the cells—is more difficult to observe with yeast.
Use the scalpel to cut the liver into very small pieces, and transfer the pieces to the saucer.
Add about 10 mL of normal saline solution to the saucer, and use the teaspoon to grind and mash the bits of liver to produce a suspension of liver cells in the saline solution.
Place a clean test tube in the rack, with the funnel atop it. Fold and refold a small piece of cheesecloth to provide four layers. Place the cheesecloth in the funnel and pour the solution from the saucer through the cheesecloth into the tube to filter out most of the solid material.
Use the stirring rod to transfer one drop of the liver cell suspension to a microscope slide and position a coverslip over the specimen. Observe the specimen at low, medium, and high-dry magnifications. Record your observations in your lab notebook. Include a sketch of a representative liver cell and its nucleus.
Add one drop of the mixed stains at the edge of the coverslip, and use the corner of a paper towel to draw the stain under the coverslip. Observe the specimen at low, medium, and high-dry magnification. Record your observations in your lab notebook. Include a sketch of a representative liver cell and its nucleus.
Use a pipette to transfer 0.5 mL of 10% sodium dodecyl sulfate (SDS) solution to the liver-cell suspension tube, and swirl gently to to mix the solutions.
Repeat steps 9 and 10 to stain and view the liver cells in the presence of the SDS solution.
Transfer another 0.5 mL of the 10% SDS solution to the liver-cell suspension and swirl gently to mix the solutions.
Repeat steps 9 and 10 to stain and view the liver cells in the presence of the SDS solution.
Repeat steps 13 and 14 several times (about five repetitions should do it) and observe the liver cells in the presence of increasing concentrations of SDS. As the cell membranes break down (are lysed) and no longer surround the nucleus, the integrity of the cells is destroyed and the nucleus and other cell contents are exposed.
Using a pipette, transfer about 4 mL of the liver-cell suspension to a second test tube. Retain the excess liver-cell suspension in the first test tube until you complete the next step successfully.
Holding the second tube at a slight angle, very slowly and carefully trickle cold isopropanol into the tube until the liquid level reaches 2 to 3 cm from the top of the tube. The goal is to cause the isopropanol to form a separate layer on top of the liver-cell suspension. If you pour too fast, the layers will mix and you’ll have to start over.
Place the tube in the rack and observe the interface between the liver-cell suspension layer and the isopropanol layer. You’ll see a cloudy whiteness begin to form at the interface. That whiteness is DNA precipitating out of solution.
Carefully and gently dip the stirring rod into the test tube until it penetrates both layers. Slowly spin the stirring rod between your thumb and finger to spool the DNA precipitate onto the stirring rod. Continue until no more DNA is deposited on the stirring rod. Withdraw the rod from the test tube.
Place one drop of distilled water on a microscope slide. Use a toothpick to remove a tiny amount of the DNA from the stirring rod. Transfer the DNA to the water drop and position a coverslip over the specimen. Add one drop of methylene blue at the edge of the coverslip, and use the corner of a paper towel to draw the stain under the coverslip. Observe the DNA at low, medium, and high-dry magnification. Record your observations in your lab notebook, including a sketch of the stained DNA.
If you want to retain the DNA you just isolated for future use, allow it to dry and then store it in a paper bag or similar porous container.
Review Questions
Q1: What was the result of adding the mixed stains to the liver-cell suspension?
Q2: Methylene blue stains acidic cell structures but leaves basic cell structures unstained. Based on that information, what do you conclude about cell nuclei?
Q3: What did you observe as you increased the amount of SDS solution you added to the liver cell suspension?
Q4: How did the appearance of the DNA differ before and after staining with methylene blue? What do you conclude about the acidity or basicity of DNA?
Q5: What do you conclude about the relative solubilities of DNA in water and isopropanol? Propose a reason why we chilled the isopropanol.