CONCEPT DEVELOPMENT #4
DNA EXTRACTION
Procedures for DNA Extraction:
The Question: How does UV Radiation play a role in genetic mutations?
Background information: Students often have trouble understanding the properties of DNA. The extreme length of DNA compared to its thickness makes it appear flexible on the macroscopic level while it is actually stiff and brittle at the microscopic level. In this lab students actually extract and handle DNA, which should reinforce your discussion of DNA structure and function. DNA is extracted from bacterial cells using liquid detergent, and then is precipitated out in alcohol. (Before undertaking this lab students need to understand the importance of sterile technique.) This inexpensive lab reinforces discussions in the areas of enzymes, cell structure, lipid membranes, supercoiling, denaturation, solubility, viscosity, detergent/lipid interaction, macromolecules (polymers) and the nature of the bacterial genome.Before beginning this activity. When working with hot water baths and glassware, students should always use caution.
1. Set up the bacteria cultures. Use sterile technique to plate out E.coli before the lab or have your students plate out their own colonies.2. Prepare a 50% solution of Palmolive dishwashing liquid in water. (Approximately 5 ml per student team).
3. Using sterile technique, the students will add one large scrape of E.coli to tubes of Luria broth (or any suitable nutrient broth). Each team of students should inoculate one tube. Incubate these suspensions overnight at 37,C (or for three days with frequent shaking at room temperature). This will provide sufficient numbers of cells for a visible mass of DNA.
Immediately before the next class, shake these cultures (to re-suspend the cells).
(Alternately, you could inoculate a large quantity of Luria broth with an equally large sample of bacteria, incubate as above and dispense these "mature" cultures in individual student aliquots. The students then will begin with student procedure step 2, and only one period would be required to perform the lab.)
When the students are ready to extract the DNA, they will need a 65-75° C hot water bath. The temperature range is critical: 60° C denatures enzymes and 80° C denatures DNA. (For this step, I use a large kitchen pot, a thermometer and a hot plate. The only real requirement is to get a volume of water large enough to prevent the temperature from dropping below 60° C during the denaturing process.)