Name:
x
Date:
Variables Affecting DNA Extraction
INTRODUCTION
Living things must be able to reproduce, and
this is done by transferring DNA from parent cells to
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offspring cells. Studying DNA is one way scientists are
able to determine what is necessary for life. In order
to analyze DNA it has to be isolated from the cell. The
purpose is to separate DNA that is present in the
nucleus of the cell from other cellular components. To
extract the DNA, scientists break open the cells and remove all of the structural proteins
and any other enzymes that interact with the DNA structure. Recall that cell membranes
have unique structural properties and are composed of lipid molecules assembled in a
bilayer.
It is beneficial to isolate DNA because it is needed for genetic analysis, which is then
used for scientific, medical, or forensic purposes. In addition, DNA can be introduced into
cells of animals and plants. Furthermore, in forensic science it’s important to extract DNA
because it can be used for identification of suspects in crime scene investigations or even
for paternity testing. Pure samples of DNA can also be used to test a newborn baby for a
genetic disease or study a gene involved in cancer. However, the samples you will collect in
this lab are not as pure as the ones used for scientific, medical or forensic purposes.
In this experiment, the cells from a banana will be chemically treated to break the
cell and nuclear membranes. The part of the cell mixture containing the DNA will then be
separated from the cell membranes and other proteins and the DNA will be “precipitated”
out and observable to the naked eye. You will also examine and analyse how the following
variables affect DNA extraction: salt, detergent, and isopropyl alcohol.
DNA EXTRACTION IS A 5-STEP PROCESS
1.
2.
3.
4.
5.
Cell lysis : Breaking the cells open
Disrupting membrane lipids with the use of a detergent or surfactant
Removing proteins with the addition of a protease (enzyme)
Removing RNA by adding an RNase
Using an alcohol to precipitate (isolate) the DNA
PRE-LAB QUESTIONS
1. What is the full name of DNA and what is its function?
x
Name:
x
Date:
2. Where is DNA located in prokaryotes and eukaryotes?
3. What is the purpose of the following reagents and how do they interact with DNA?
i. Dishwashing liquid
ii. Isopropanol
iii. Salt
4. What is the purpose of crushing the banana into a pulp?
5. Would this protocol work on food samples that are cooked or heated? Why or why
not?
6. Why would we need to separate the DNA from the proteins and lipids?
7. What is the purpose of having a control?
x
Name:
x
Date:
TESTING EXPERIMENTAL VARIABLES
You will be given a specific variable to test in this experiment. You will only change
the amount/temperature of ONE variable; the amounts are given in the chart below. The
data that you collect will be pooled into a class data, and you will generate a graph to show
a visual representation of how the different variables affect the quantity of DNA extracted
compared with the control samples. Check the box of the variable that you and your group
will be testing. In steps 1 and 8 of your experimental procedure, fill in the appropriate
amounts for the reagents, taking into account the variable you will be analysing.
My Experimental
Variable
Variable
Amount/Temperature
Salt
Detergent
Isopropyl Alcohol
½ spoon
2 spoons
Room Temperature
PREDICTION: How do you think your experimental variable will affect the amount of DNA
extracted?
SAFETY
Materials used are safe to handle with bare hands.
Ensure that alcohol is not near a source of open flame.
If glass is broken, use a brush and broom to dispose of shards into the glass box. Do not use
bare hands.
DISPOSAL
All reagents and products of experiment can be safely disposed of in the garbage.
MATERIALS
Distilled Water
Chilled 70% isopropyl alcohol
25 ml graduated cylinder
2 100 ml beakers
2 50 ml beakers
2 glass stirring rods
2 coffee filters
Table salt
Detergent
½ banana
Plastic resealable bag
2 plastic spoons (teaspoon)
Electronic balance
Parchment paper
GENERAL PROCEDURE FOR CONTROL AND EXPERIMENTAL
Place banana in plastic bag, remove air, and seal. Squash banana until there are no solid
pieces remaining. You will use this banana slurry for both your control and experimental
samples.
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Name:
x
PROCEDURE for control sample
1.
2.
3.
4.
5.
6.
7.
8.
Date:
x
NOTES/OBSERVATIONS
Prepare your extraction buffer. To a 50 ml beaker,
add:
o 15 ml of water
o ¼ spoon of table salt
o 1 spoon of detergent
Mix well using the stirring rod, but be careful not to
create too many bubbles.
To a 100 ml beaker, add:
o 2 spoons of the banana slurry
o all of the extraction buffer
Mix well for approximately 4-5 minutes using the
stirring rod, being careful not to create too many
bubbles. During this time, rinse the 50 ml beaker free
of detergent.
Fold the coffee filter into a cone-like shape and place
it into the clean and labelled 50 ml beaker. Do not let
the filter paper touch the bottom of the beaker.
Pour the banana mixture from the 100 ml beaker
into the coffee filter to collect your filtrate. You
should collect 10 ml of filtrate for sufficient DNA.
After you have obtained banana filtrate, you can
dispose of the filter paper and banana remains in the
garbage.
In your graduated cylinder, obtain chilled 70%
isopropanol. Pour an equal volume (10 ml) of the
alcohol into the beaker with your banana filtrate.
Swirl the mixture gently, and let settle for 2-3
minutes. You should see DNA precipitating out of the
alcohol layer where the two phases meet.
9.
Pre-weigh your square of parchment paper, and label
it with your name and sample type.
10. Use your stirring rod to pick up the DNA by slowly
swirling the rod.
11. Spread your DNA out on the parchment paper. Let
the DNA sit for a few days until it is completely dry.
Weight of paper:
12. Weigh the filter paper with DNA and determine the
amount of DNA extracted.
Weight of paper + DNA:
Weight of DNA:
g
g
g
Name:
x
PROCEDURE for experimental sample
1.
2.
3.
4.
5.
6.
7.
8.
Prepare your extraction buffer. To a 50 ml beaker,
add:
o 15 ml of water
o
spoon of table salt
o
spoon(s) of detergent
Mix well using the stirring rod, but be careful not to
create too many bubbles.
Date:
x
NOTES/OBSERVATIONS
Indicate the variable you are testing:
How is the variable altered?
To a 100 ml beaker, add:
o 2 spoons of the banana slurry
o all of the extraction buffer
Mix well for approximately 4-5 minutes using the
stirring rod, being careful not to create too many
bubbles. During this time, rinse the 50 ml beaker free
of detergent.
Fold the coffee filter into a cone-like shape and place
it into the clean and labelled 50 ml beaker. Do not let
the filter paper touch the bottom of the beaker.
Pour the banana mixture from the 100 ml beaker
into the coffee filter to collect your filtrate. You
should collect 10 ml of filtrate for sufficient DNA.
After you have obtained enough banana filtrate, you
can dispose of the filter paper and banana remains in
the garbage.
In your graduated cylinder, obtain chilled/unchilled
70% isopropanol. Pour an equal volume (10 ml) of
the alcohol into the beaker with your banana filtrate.
Swirl the mixture gently, and let settle for 2-3
minutes. There will be 2 layers; you should see DNA
precipitating out of the alcohol layer where the two
phases meet.
9.
Pre-weigh your square of parchment paper, and label
it with your name and sample type.
10. Use your stirring rod to pick up the DNA by slowly
swirling the rod.
11. Spread your DNA out on the parchment paper. Let
the DNA sit for a few days until it is completely dry.
Weight of paper:
12. Weigh the filter paper with DNA and determine the
amount of DNA extracted.
Weight of paper + DNA:
Weight of DNA:
g
g
g
u
u
Name:
x
Date:
CLASS DATA
Create a chart below to record the class data. You will determine the average mass of DNA
for all of the controls, and for each of the variables.
x
Name:
x
Date:
POST-LAB ANALYSIS QUESTIONS
1. Describe and draw the appearance of the DNA you extracted. Make sure to include
colour, shape, size, and consistency.
2. How do the results of your experimental sample compare with your prediction? Does
your data support or refute your initial prediction?
3. What could you do differently to increase the yield and purity of the DNA sample? What
other reagents might be of use?
x
Name:
x
4. What were some sources of procedural error?
Date:
5. If you try to extract DNA from the same amount (by weight) of grapes, would you obtain
more or less DNA? Explain.
6. Compare and analyse the results of your graph. Explain, on a molecular level, how the
amounts used for each of these variables affected the amount of DNA extracted.
x