1991 Woodrow Wilson Biology Institute
At the end of this activity the student should be able to:
- Extract and observe a visible amount of DNA from Escherichia. coli cells.
- Discuss the properties of DNA.
- Confirm the presence of DNA by a reaction with an indicator.
The age-ability level(s) for this activity is grades 7-l2 and most ability level.
Life involves complex chemical interactions that take place among the many kinds of molecules found with in cells. Deoxyribose Nucleic Acid (DNA) is the master molecule, the structure of which encodes all information needed to create and direct the chemical machine of life. Different patterns in the DNA molecule produce the diversity found in all living organisms.
This hands-on activity provides an opportunity for students to use a simple technique to extract bacterial DNA. DNA is found in the cells of all living organisms, including man. However, it is easier to extract DNA from a bacterium because the DNA is not enclosed in a nuclear membrane. Since the structure of DNA is similar in all organisms, extracting bacterial DNA provides a model for understanding properties of human DNA. This activity may be used with the following biology units: Unity and diversity of life, carbon compounds in cells, cell structure and function and human genetics.
- E. coli colony from agar plate
- l inoculating loop
- 2-l5 ml culture tubes
- 20 ml Luria broth (or regular nutrient rich broth)
- incubator @ 37 oC (optional)
- l0 ml solution (50%) dish washing detergent in water
- l hot plate
- l 500 ml beaker
- l thermometer ( C)
- l eye dropper
- l0 ml ethanol (95%)
- l stirring rod
- Obtain individual E. coli colonies from a culture plate. Use a sterile inoculationg loop to suspend a 2-3 mm mass of E. coli in a l5 ml culture tube containing 5 ml of Luria broth. Label this tube A. Add 5 ml of Luria broth to another culture tube without adding any E. coli. Label this tube B. Incubate both tubes overnight at 37 C or over 3 days at room temperature.
- Add to both labeled tubes 3 ml of a 50% solution of dishwashing detergent in water. Shake each tube to assure complete mixing. Shake the suspension to rupture (lyse) cells and release DNA.
- Prepare a 65-75 oC water bath. Place each tube into a water bath for l5 minutes. Maintain water bath temperature accurately. A temperature greater than 60 oC is needed to destroy enzymes, but temperatures greater than 80 oC will denature (break apart) DNA.
- Use a dropper to add 5 ml of cold 95% ethanol onto the lysed cell suspension in each tube. Water-soluble DNA is insoluble in ethanol, and a white, web-like mass (precipitate) of DNA will float in the alcohol. Gently spool the DNA onto the rod with a twisting motion.
- A diphenylamine indicator will confirm the presence of DNA.
- a. Label three test tubes l, 2, and 3.
- b In test tube l, add 3 ml of sodium chloride solution (4%) (2 grams of sodium chloride to 50 ml of distilled water) and 3 ml of crude (extracted) DNA; add 3 ml of diphenylamine indicator and mix.
- c. In test tube 2, add 3 ml of DNA standard solution; add 3 ml of diphenylamine indicator and mix.
- d. In test tube 3, add 3 ml of distilled water; add 3 ml of diphenylamine indicator and mix.
- e. Place tubes in boiling water for l0 minutes and record the color changes. Diphenylamine indicator reacts with deoxyribose compound of DNA to produce a blue color.
Discussion topics from this activity:
- What is DNA?
- What is the structure of DNA?
- What are the properties of DNA?
- What is the function of DNA?
- What is species diversity?
Bost, Rod l989. Down and Dirty DNA Extraction. Carolina Genes. Research Triangle Park, N.C., North Carolina Biotechnology.
Alleghany High School
Sparta, North Carolina 28675