Extraction of DNA from Onion

Jo Ann Lane
1994 Woodrow Wilson Collection

Overview
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.
Objectives
1. To become familiar with the physical properties of DNA by isolating it from living tissue
2. 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:
• blender
• 60û C water bath
• thermometer
• 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 cheesecloth (4 thicknesses
 2-100 ml graduated cylinders (one kept on
• cutting board ice)
• medium onion  250 ml beaker
• knife  500 ml beaker
 1000 ml beaker
• weighing boat  glass stirring rod
• funnel

DNA Isolation Procedure

Homogenization:
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.
1. 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.
2. Weigh out 50 g of diced onion. Transfer all of the weighed material to a 250 ml beaker.
3. 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.
4. 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.
5. 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).
6. Pour the homogenate from the blender into a 1000 ml beaker. Allow it to stand in an ice
bath for 15-20 minutes.
7. 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.
8. Place your beaker with its filtered homogenate into an ice bath. Let it cool until it reaches
10-15û C (about 10-15 minutes).
9. 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.
 10. 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.
11. 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.
Questions
1. Why is it important to rotate the rod in the same direction when spooling the DNA onto
the rod?

2. What did you learn about the properties of DNA during this laboratory period?

3. 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.)

Teacher Information
1. 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
2. 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.
3. 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.
4. 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.
5. Stress with the students that they must follow the directions carefully since the
temperatures and timings are crucial to the procedure.
6. 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.
7. You may have some small vials and 50% ethanol available so that the students may save
their DNA.
8. To save time you may use a blender to chop up the onions and dispense them in 50 g
portions.
9. There is a lot of "waiting time" in this lab, so a worksheet on DNA can be completed
during this time.
10. Wear a mask when massing the sodium laural sulfate-it is very powdery and gets into the
air.