Extraction of DNA from Onion
Jo Ann Lane
1994 Woodrow Wilson Collection
To most students of biology, DNA is an abstraction. You can memorize the names and structures of the nitrogenous bases and know all about the history of DNA's discovery, but until you actually handle DNA, it remains a strange and mysterious substance.
The purpose of this laboratory is to give you firsthand experience with DNA by isolating it from plant tissue. You will start with whole onions and end with a relatively pure preparation of DNA, containing literally millions of genes. Once isolated, the DNA can be stored in alcohol or dried out. It will actually be possible for you to hold in your hands the key to an organism's development and structure.
- To become familiar with the physical properties of DNA by isolating it from living tissue
- To learn the purpose of each step in the isolation procedure as it relates to the physical and biochemical characteristics of the genetic material
Materials and Equipment
The following materials can be shared by a group of students:
- 60û C water bath
- ice bucket
- balance (0.1 g scale)
- 95% ethanol kept on ice
The following materials are needed for each student or pair:
- plastic gloves
- 100 ml homogenization medium
- cutting board
- medium onion
- weighing boat
cheesecloth (4 thicknesses
2-100 ml graduated cylinders (one kept on ice)
250 ml beaker
500 ml beaker
1000 ml beaker
glass stirring rod
DNA Isolation Procedure
Before DNA can be released from the nuclei of the onion tissues, the cell walls, plasma membranes, and the nuclear material must first be broken down. This step can be accomplished by homogenizing the onion tissues in a blender. The detergent solution causes the cell membrane to break down and emulsifies the lipids and proteins of the cell by disrupting the polar interactions that hold the cell membrane together. The DNA can then be separated from the chromosomal proteins by the chemical components of the homogenizing medium which will cause the proteins to precipitate out of solution.
- Wearing plastic gloves, dice a medium-sized onion into cubes no larger than 3 mm. (This step may have already been done for you to save time.) Plastic gloves prevent DNAse enzymes on your hands from cutting the DNA into small fragments so that it will not spool.
- Weigh out 50 g of diced onion. Transfer all of the weighed material to a 250 ml beaker.
- Add 100 ml of homogenizing medium to the diced onion and incubate the beaker in a 60û C water bath for 15 minutes (no longer!). This heat treatment softens the onion tissue and allows penetration of the homogenization solution. It also denatures many enzymes that could interfere with the isolation procedure.
- Quickly cool your preparation to 15-20û C in an ice bath (a slush of ice and water). This step should be accomplished in about 6 minutes and prevents the denaturation of DNA.
- Pour your cooled preparation into a blender and fasten the lid. Homogenize for 45 seconds at low speed, followed by 30 seconds at high speed. Homogenization breaks open the cells and releases their contents (carbohydrates, proteins, fats, and nucleic acids).
- Pour the homogenate from the blender into a 1000 ml beaker. Allow it to stand in an ice bath for 15-20 minutes.
- Filter the homogenate through four thicknesses of cheesecloth into a 500 ml beaker, taking care to leave the foam behind.
Precipitation of DNA:
The homogenate should contain only DNA and the components of the homogenizing medium. Of the components remaining in the homogenate, only DNA is not soluble in ice-cold ethanol. Therefore, when ice-cold ethanol is added to the homogenate, all the components of the homogenizing medium stay in solution-except DNA. If the instructions have been followed carefully so that the molecular structure of DNA remains intact, the genetic substance should precipitate as a thick, stringy, white mass that may be spooled out by winding it on a glass rod. If the DNA has been damaged, it will still precipitate, but as a white, fuzzy mass that cannot be collected on a glass rod.
- Place your beaker with its filtered homogenate into an ice bath. Let it cool until it reaches 10-15û C (about 10-15 minutes).
- Measure out 80 ml of ice-cold ethanol into a cold graduated cylinder. Slowly add the ethanol down the side of your beaker until the white, stringy DNA precipitate appears. It may not take all 80 ml of the alcohol to precipitate your DNA.
- Spool out, or wind up, the stringy DNA onto a glass rod by rotating the rod in one direction only in the beaker of DNA. Continue to rotate the rod as you move it in large circles through the beaker.
- If you want to keep your DNA, gently ease it off the end of the glass rod into a vial filled with 50% ethanol. Be sure the cap is tight enough to prevent leakage.
- Why is it important to rotate the rod in the same direction when spooling the DNA onto the rod?
- What did you learn about the properties of DNA during this laboratory period?
- What structural characteristics of DNA allows it to be spooled out on a glass rod? Why is it not possible to spool out precipitated proteins? (Hint: Compare the relative lengths of DNA and protein molecules.)
- Preparation of homogenization medium:
|sodium laural sulfate (SDS or SLS) ||50.000 g|
|sodium chloride ||8.770 g|
|sodium citrate ||4.410 g|
|ethylenediamine tetraacetic acid (EDTA) ||0.292 g|
Add distilled water to make 1 liter of solution. Do not place in refrigerator or the SDS will turn the solution an opaque white color. If the homogenization medium gets cold at any time, it will turn white, but this will not affect its function.
- Ethanol must be cold for this procedure to work. Place a bottle of ethanol and one graduated cylinder for each lab group in a freezer overnight. Be sure the cap is loose and the bottle is not completely full. Place the bottle on paper towels. It will not freeze. Just before lab dispense the ethanol into smaller containers and put on ice. Or you can fill one smaller bottle for each lab group and pass out the bottles and graduated cylinders directly from the freezer or from a cooler filled with ice just before the students will use them.
- Have the students wear gloves and tell them not to touch the inside of containers because DNAse enzymes from their hands will break the DNA into small fragments so that it will not spool at the end of the lab. Rinse all glassware with distilled water.
- Stress with the students that they must follow the directions carefully since the temperatures and timings are crucial to the procedure.
- Scoring the end of the glass rods with sandpaper will help the DNA adhere to the rod while spooling. Do not touch the end of the rod with your fingers.
- You may have some small vials and 50% ethanol available so that the students may save their DNA.
- To save time you may use a blender to chop up the onions and dispense them in 50 g portions.
- There is a lot of "waiting time" in this lab, so a worksheet on DNA can be completed during this time.
- Wear a mask when massing the sodium laural sulfate-it is very powdery and gets into the air.