||The purpose of this lab is to attempt to Isolate DNA
from Strawberries. In doing so you will experience some of the steps
taken by scientists to acquire DNA which could be used in a variety
of experiments. The Human DNA analyzed as part of the Human Genome
Project was isolated in a similar way. If the amount of DNA recovered
is extremely small, say perhaps from a crime scene or from an extinct
life form, many millions of copies must first be made of that which
was recovered by using PCR technology. Having millions of copies would
be needed to run experiments that will reveal the makeup of the DNA
collected.We will talk about the process of PCR technology at a later
time. For now.....let's isolate!
||Small ziplock bag
Make Sure ALL Glassware is SQUEKY CLEAN before mixing any reagents.
1. Place 1-2 large strawberries into a small ziplock bag and crush the
berries until pulpy (about 1-2 minutes). Have the other member of
your lab team make the lysis buffer while the strawberry is being crushed.
2. Make the lysis buffer in a cup by combining
- 10 mL of water
- 1⁄2 Teaspoon Dish soap
- 1⁄2 Teaspoon salt
3. Place 2 Teaspoons Lysis Buffer into the bag and mix for about a minute.
4. Pour the strawberry mixture into a funnel lined with paper towel into
a test tube and SQUEEZE the mixture into the tube. Make sure that all
strawberry extract passes through the paper towel before entering the
test tube. This will insure that large particles are strained out of extract.
5. Add an equal or slightly greater volume of rubbing alcohol into the
test tube to at least double the volume of the strawberry mush.
6. Place a bamboo skewer into the tube and watch very carefully as you
SWIRL the stick.
7. Show me your results.
Questions:(use the web for help
- What is a lysis buffer?
- What was the purpose of including dish soap in the lysis buffer?
- Why was rubbing alcohol added to the test tube with the strawberry
- Briefly describe what the DNA looks like in your test tube.
- What purpose does the bamboo skewer serve?
- If your DNA sample was tested for the presence of Nitrogen what would
the results be? Explain your answer.
- Using the web for help briefly describe how a PCR
machine (aka: thermal cycler) works.
- What are restriction enzymes?
- What is meant by Recombinant DNA?
- Why would "sticky
end" cutting restriction enzymes be used when attempting to
move DNA from one organisms to another?
- Why must the same "sticky end" cutting restriction enzyme
be used on both sources of DNA when attempting to create Recombinant
- When a large amount of DNA is mixed with restriction enzymes RFLPs
are formed. What is meant by RFLP?
- What biotech protocol is used to seaparate RFLPs?
- How does gel
electrophoresis allow for the creation of a DNA
- You are a CSI Investigator and you have collected tissue from underneath
the fingernails of a crime victim. Detectives have apprehended two individuals
who both had motive and no alliby. Briefly describe the steps you would
take (in correct order) to determine which of the suspects (if any)
may have commited the crime.
Visit the following
web site and check your understanding of how to make a DNA fingerprint.
Cheek Cell DNA Extraction
Individual strands of DNA are too small to be visible to the eye. One
million threads of DNA fit onto the period at the end of a sentence using
Times New Roman, font 12 in WORD. The reason why we are able to see DNA
in this activity is that there are so many of them, clumped together.
DNA extraction is a fairly simple procedure that requires only a few
steps: 1. The detergent breaks open the cells by destroying the fatty
membranes that enclose the cells as well as the nuclei membranes within
the cells. DNA is released into the solution. Detergent and the salt
also helps strip away proteins that are associated with the DNA molecules.
2. DNA is NOT soluble in alcohol, whereas other cell parts are. By adding
alcohol, DNA precipitates out of the solution and collects at the interface
of the alcohol and soap layer. The colder the alcohol, the less soluble
the DNA will be in it.
BE SURE TO WASH HANDS BEFORE AND AFTER THIS EXERCISE. BE VERY
CAREFULL NOT TO ACCIDENTALLY SHARE SOLUTIONS.
- A 0.9 percent salt solution (2 teaspoons of table salt
dissolved in 1 quart/liter of water) is in a Alhambra Jug in the back
of the room.
- Pour saline solution in one of the small plastic cups appx 1/6 full
(see image on white board)
- Swirl the 10ml of salt solution in your mouth for 30 seconds. This
will remove dead cells lining the mouth.
- Carefully spit solution back into the small plastic cup and then
pour as much of the solution as is possible it into the reaction tube.
Pour off excess into the sink. Flush the sink with water and be sure
to throw the plastic cup into the trash.
- Bring reaction vessel to me at the centrifuge 20 minutes before the
- Collect your reaction vessel and look at the very bottom for a small
ball of cells.
- Carefully pour off as much of the content WITHOUT DISRUPTING OR LOSING
THE BALL OF CELLS.
- Prepare 25 percent detergent solution (1
drop detergent 3 drops of water)
- Add detergent solution into plastic reaction
- Gently rock reaction vessel back and forth for 30 seconds to a minute.
DO NOT SHAKE VESSEL VIGOROUSLY. THIS WILL BREAK UP THE DNA AND MAKE
IT HARDER TO SEE.
- Fill the remainder of the vessel with rubbing alcohol using your
- The alcohol and the detergent should form two distinct layers with
the alcohol sitting on top.
- Let the tube stand for one minute and then use cofee strirrer to
slowly move some of the ethanol into the soap layer. DNA will start
to precipitate out of the soap solution. Twirl the stirrer
to spool the DNA strands around it.
- If you wish to take your DNA home come to me for a new reaction vessel
to transfer the DNA into.