Physiology Lab
p1/7
Name:___________________________________
Extraction of DNA from Onion
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:
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52 ºC water bath
thermometer
ice bucket
0.1 g scale
95% ethanol kept on ice
The following materials are needed for each student or pair:
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plastic gloves
100 ml homogenization medium
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cutting board
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medium onion
knife
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weighing boat (plastic cup)
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cheesecloth (4 thicknesses
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500 ml beaker (kept on ice)
2-100 ml graduated cylinders (one kept on ice)
250 ml beaker (kept on ice)
1000 ml beaker
DNA spooling rod
Physiology Lab
p2/7
Name:___________________________________
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 gently mashing the chopped onions while in the water bath. 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.
Wear gloves and do not 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. Do not touch the end
of the rod with your fingers.
You must follow the directions carefully since the temperatures and timings are
crucial to the procedure.
1. Chill the 250 ml beaker by sitting it in an ice bath while doing the following:
2. 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.
3. Weigh out 60 g of diced onion. Transfer all of the weighed material to a 1000 ml
beaker.
4. Add 100 ml of homogenizing medium to the diced onion and incubate the beaker
in a 52 ºC water bath for 15 minutes (no longer!). Using a mortar pedestal or
spoon crush the onion against the sides and bottom of the beaker while it is in the
water bath. 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.
5. Quickly cool your preparation to 10-14 ºC in an ice bath (a slush of ice and water
more ice than water). This step should be accomplished in about 6 minutes and
prevents the denaturation of DNA. Continue to crush the onion against the beaker
6. Filter the homogenate through four thicknesses of cheesecloth or through a # 6
coffee filter into a chilled 500 ml beaker, taking care to leave the foam and solid
material behind.
7. Pour the filtered homogenate into a chilled 250 ml Beaker.
Physiology Lab
p3/7
Name:___________________________________
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 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 spooling rod. If the DNA has been damaged, it will still precipitate, but as a
white, fuzzy mass that cannot be collected on a collecting rod.
8. 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.
9. Spool out, or wind up, the stringy DNA onto a collecting 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.
10. 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.)
Physiology Lab
p4/7
Name:___________________________________
Preparation Information
1. Measure 50 ml liquid detergent into a 1000 ml beaker. Add distilled water to
make 750 ml of solution. Add 11.25 grams NaCl. Refrigerate
2. 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.
3. 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.
4. Stress with the students that they must follow the directions carefully since the
temperatures and timings are crucial to the procedure.
5. 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.
6. You may have some small vials and 50% ethanol available so that the students
may save their DNA.
7. To save time you may use a blender to chop up the onions and dispense them in
50 g portions.
8. There is a lot of "waiting time" in this lab, so a worksheet on DNA can be
completed during this time.
Physiology Lab
p5/7
Name:___________________________________
Watch the Chromosome Flyover movie at http://www.dnalc.org/ddnalc/resources/chr11.html
1. Variations in the number of repeats between people in the Intergenic Region can be
used for:
Go to http://www.dnalc.org/ddnalc/resources/shockwave/dnadetective.html How to
make a DNA fingerprint/ more/ more/ continue …..
2. DNA is extracted from samples of blood, semen or hair roots. Then the DNA is
cut up into a precise set of fragments with ___________________
__________________.
3. The agarose gel acts as a molecular sieve to
___________________________________________________________
Continue with the Florida Murder Rape Case
4. How does the comparison of the semen sample and the DNA taken from the
suspects show positive identification of the rapist? How does it exclude the other
suspect from being the rapist?
5. What was the chance that another person would have the same DNA fingerprint?
6. What made this case a historical case?
Continue with the Human Paternity Case
7. In the Human Paternity case, why did not the DNA match show conclusively that
the child was born of the alleged mother.
Physiology Lab
p6/7
Name:___________________________________
8. The father of the uncontested children’s DNA was reconstructed from the DNA
pattern from the uncontested children by producing a pattern matching those of
the children that were not contained in the mother’s DNA. How did this help the
case?
9. What was the probability that the child could have been born of only that father
and mother combination?
Continue with the Horse Paternity Case
10. In the horse paternity case, did the father or the son sire the foal, and how do you
know from the DNA information?
Go to http://www.pbs.org/wgbh/nova/sheppard/lab01.html
Read Part 1 and Click on Create a DNA fingerprint. / It takes a Lickin’. Follow the story
line and continue with Part 2.
Click and drag the restriction enzymes over the DNA and move about until they dump
into the DNA.
11. Under What Happened where does the example restriction enzyme cut DNA.
12. Pour the agarose gel into the tray. How does the agarose work to separate the
fragment sizes?
13. Electrophoresis is the process of ___________________________________ with
an electric current.
14. What purpose does the nylon membrane serve?
Physiology Lab
p7/7
Name:___________________________________
15. The X-Ray film is exposed by:
16. Who desecrated Jimmy Sweet’s lollipop, and how do you know?