Download Kiwi DNA Extraction Activity

Lesson 5: DNA Extraction Lab
Introduction
DNA is present in the cells of all living organisms. This
procedure is designed to extract DNA from kiwi in
sufficient quantity to be seen and spooled. Why is it
necessary to extract DNA out of a cell in genetic
engineering? In order for genetic engineers to be able
to work with DNA and transfer it into another organism,
it must be first taken out of the cell. Fortunately, DNA
remains somewhat stable outside of a living cell,
allowing scientists the opportunity to work with and
study the cell without destroying it.
Purpose:
State a purpose for this lab activity.
Materials:
- two 1-cup measuring cups or beakers (250 mL) with
mL markings
- measuring spoons
- knife for cutting kiwi
- large spoon for mixing and mashing kiwi
- thermometer that can measure 60°C (140°F)
- strainer that will fit in the 1-cup measuring cups
- hot tap water (60°C) (a 3L saucepan works well to
hold the water)
- hot plate
- distilled water
- green dish soap
- 1 kiwi
- table salt, either iodized or non-iodized
- 1 test tube for each student, preferably with a cap, to
hold the kiwi DNA solution.
- 99% ethanol (available at a pharmacy as "rubbing
alchohol") kept in freezer overnight
Procedure:
1. Set up hot water bath at 55-60°C.
2. For each kiwi, make a solution consisting of 10 mL
(one tablespoon) of liquid dishwashing detergent and
1.5 g (one level 1/4 teaspoon) of table salt in a 250-mL
beaker (1-cup measuring cup). Add distilled water to
make a final volume of 100 mL. Dissolve the salt by
stirring slowly to avoid foaming. Avoid making bubbles.
3. Peel the kiwi and cut it into about 12 pieces. Then
add the pieces to the solution made in step 2 being
kept in the measuring cup or beaker.
The detergent causes the cell membrane to break
down by emulsifying the lipids and proteins of the cell
and disrupting the polar interactions that hold the cell
membrane together. The detergent then forms
complexes (micelles) with these lipids and proteins,
causing them to precipitate out of solution.
The salt shields the negative phosphate ends of the
DNA that allows these ends to precipitate out of a cold
alcohol solution.
4. Put the measuring cup or beaker in a hot water bath
(don't put the solution itself in the bath!) for 10-12
minutes. Keep the temperature of the bath consistently
55-60°C. During this time, press the kiwi against the
sides and corners of the measuring cup with the back
of the spoon. Do not keep the mixture in the hot water
bath for more than 15 minutes because the DNA will
break down (or denature).
The heat denatures the DNAase enzymes
(endonucleases) that have the potential to break down
genomic DNA into tiny pieces and prevent DNA from
spooling.
5. Filter the mixture through a strainer into your second
measuring cup or beaker. When pouring the mixture
into the strainer, avoid letting foam get into the
measuring cup.
6. Dispense the kiwi DNA solution into test tubes, one
for each student. The test tube should contain about 10
mL of solution (1 teaspoon).
7. Add cold alcohol to the test tube to create an alcohol
layer of roughly the same volume as the kiwi DNA
solution dispensed in step 6. For best results, the
alcohol should be as cold as possible (keep in freezer
overnight). When alcohol is added to the mixture, the
components of the mixture, except for the DNA, stay in
the solution while the DNA precipitates out into the
alcohol layer.
The cold slows the rate of DNA breakdown while the
alcohol acts as a precipitating factor, because DNA is
not soluble in the soap, salt, and distilled-water
solution.
8. Let the solution sit for two to three minutes without
disturbing it (that is, not shaking it). You can watch the
white DNA precipitate out into the alcohol layer. When
good results are obtained, there will be enough DNA to
spool on to a glass rod or a Pasteur pipette. DNA has
the appearance of white mucus.
Observations:
State your observations for this lab.
Interpretations:
1. Four barriers needed to be broken through to reach
the DNA inside the nucleus. How was this done for
each barrier? (Hint: Think about the structure of a plant
cell.)
2. What "ingredient" helps the DNA come out of
solution or precipitate?
3. Do you think the DNA obtained was pure? Why or
why not? Give two reasons.
4. Name the three properties of DNA that are
demonstrated by this lab.
5. Suggest 5 experimental errors that occurred with this
lab. If you are unsure of what experimental error is,
ask your teacher.
Conclusion:
State 2 conclusions from this lab.