Extracting DNA from plants and bacteria.
Scientists today estimate only about 5% of the species of microbes that live on earth can be cultured by standard methods. The other 95% of all microbes will not grow in the lab because the specific requirement for their normal growth cannot be reproduced. Some of this is due to a lack of knowledge on what their growth requirements are, and some is due to the technical difficulties in reproducing these requirements in the laboratory. Because of this, the Kartchner Caverns project relies heavily on non-culturable methods of analysis that require microbial DNA to be extracted directly from samples without the need to culture the organisms. How do we get DNA? This experiment will allow students to extract and purify DNA directly from plants and bacteria and see it actually precipitate out in a test tube! This fascinating technique is the first step used in “molecular” biology and to appreciate its importance is key to understanding the full diversity of microbes that surrounds us everyday.
What you will need:
1) a blender
2) a strainer
3) stiring rods
3) a measuring cup
4) small glass containers
5) rubbing alcohol
6) ½ cup split peas (or 100 ml)
7) 1/8 tsp table salt
8) 1 cup of water
9) detergent (liquid dishwashing)
10) meat tenderizer (or pineapple juice or contact lens cleaning solution)
DNA is the blueprint of life. In this experiment, we will extract the DNA from some plant material, like split peas, but you can use just about any plant material to extract DNA from. In addition, you can extract DNA from a culture of bacteria, such as one you might have isolated from soil in the above experiment, using very similar techniques.
Plant DNA Isolation.
- Combine peas, salt, and water in the blender and process on high speed for about 15 seconds. This will break up the peas so that they are more exposed to the action of the detergent in later steps.
- Next, take theresulting pea soup and put it through a strainer and collect the liquid portion in a separate container. Then add about 30 ml, or 2 tablespoons, of liquid detergent and mix by swirling.
Let the mixture sit at room temperature for 5-10 minutes and then transfer to individual glass test tubes or clear glass containers. Only fill the containers 1/3 of the way full because you will need room to add more reagents soon.
Next, add a pinch of the meat tenderizer (or above listed alternatives) to each tube so that it can start to break down remaining proteins in the mixture. Gently stir the solution with a sterile stirrer, but do it gently to avoid breaking up the DNA strands.
Now comes the fun part! Add an equal part volume of rubbing alcohol (70-95% isopropanol) to the tubes. Do this gently so that the alcohol forms a layer on top of the pea mixture.
The stringy white stuff you will see forming at the interface of the two liquids is DNA! You can then gently pulse the wooden end of a cotton swab, or other stirring rod, up and down through the alcohol and pea soup boundary to collect precipitated DNA. It will collect as long stringy white strands and is pure DNA!
Bacterial DNA isolation.
- Transfer a small amount of a pure bacterial culture using a cotton swab or a toothpick into a test tube containing Tryptic Soy Broth (Difco) or LB broth (Difco). Use 4 ml of media per tube and exercise STERILE TECHNIQUES when you transfer to prevent other contaminants from being introduced to your growth medium. Incubate the tubes for 48 hrs at 37C.
- Add 3 ml of a 50% dishwashing detergent to the culture and mix well.
- Incubate the tube in a 70C water bath for 15 minutes.
- Push a glass rod through the alcohol layer into the bacterial/detergent suspension, then twist and pull the rod up back into the alcohol layer just like you did for the plant DNA isolation. Repeat until you have a generous supply of "spooled" DNA on the glass rod
Now that you have pure DNA, you can transfer the DNA to a glass slide and stain with 1 drop of Aceto-Orcein to observe the fiber-like nature of the molecule.
Aceto-Orcein: Orcein 1 gm
Lactic Acid 85% 28 ml
Glacial Acetic Acid 22 ml
DNA is pretty tough and will remain in the “stringy” state for many days without degrading. It has to be tough since it contains all the instructions for life in every living cell! Once purified, molecular biologist use this same type of DNA preparation for many very complex experiments including isolation of specific genes, insertion of new DNA into existing genes, and inserting these modified genes into new organisms. These basic techniques of molecular biology have revolutionized science over the last 40 years and have led to many of the incredible discoveries we read about daily in areas of agriculture, medicine, and environmental study. DNA isolation is only the first step into the amazing world of molecular biology!