LESSONS #1-3
DNA EXTRACTION LAB
How do genotype and allele frequencies affect the likelihood of a heart attack occurring in the future?
The purpose of this lab is to collect DNA sample from the cells that line the inside of your mouth and use
this sample to determine your particular gene. We are also going to examine the genotype and allele
frequencies of the class and compare it to the other Biology (Engineering Pathway) class.
This lab utilizes (uses) many lab techniques, including using micropipettes, PCR amplification and gel
electrophoresis. You will have hands on experience with a micropipette and analyzing photos from gel
electrophoresis.
Micropipettes are an important tool used in genetic engineering. A
micropipette is used to transfer very small and exact volumes of
liquids in either milliliters (mL, thousandths of a liter) or microliters
(μL, millionths of a liter).
Polymerase Chain Reaction (PCR) has many applications. It is
commonly used to produce many copies of a selected gene segment
or locus of DNA. In criminal forensics, for example, PCR is used to
amplify DNA evidence from small samples that may have been left at
a crime scene. A skilled technician can even obtain a DNA sample left
by the tongue on the back of a postage stamp used to send a letter .
Gel electrophoresis is used to separate and identify a mixture of
biomolecules including DNA. The components of each mixture can
then be identified by their location in the gel.
Figure 1: micropipette
In this lab, the locus we will amplify is located in the tissue Plasminogen Activator (tPA) gene. In other
words, the part of the DNA we will make a lot of copies of and analyze is found in the tPA gene. The tPA
gene is carried on chromosome 8, and the gene codes for a protein that is involved with dissolving blood
clots. tPA is a protein administered to heart attack victims to reduce the incidence of strokes.
The gene that we will be targeting for amplification is dimorphic, which means the locus (that part of the
gene) has two forms. One form carries a 300 bp DNA fragment known as an Alu element, and the second
form of the locus does not carry this fragment. Therefore, when we examine this locus, we find that it
may or may not carry an Alu element.
DAY 1: HOW TO USE A MICROPIPETTE
Materials to collect:
 P-20 micropipette
 Tip box of disposable pipette
 green dye
 Laminated micropipette practice sheet
 Used tip container
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We are going to use a P-20 micropipette. A P-20 micropipette can transfer a maximum of 20.0 μL and a
minimum of 2.0 μL
Warnings:
1. NEVER use a micropipette without a plastic tip
2. NEVER use the plastic tip twice – or else you’ll contaminate samples
3. NEVER let the plunger snap back into place – slowly guide it back
4. NEVER try to measure a volume that the micropipette cannot measure – you will damage the
micropipette!
5. Always push on the plunger first, then insert the plastic tip into the liquid
6. NEVER turn the micropipette upside down with liquid in the tip
Directions:
1. Find the display window on the handle of the micropipette.
2. Make sure the display indicates 20.0 μL
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a. It should look like the picture on the right
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b. If it is not, turn the black wheel on the
0
handle to clockwise to decrease the
number or counterclockwise to increase the number
3. Press the micropipette into one of the small tips in the box.
a. Close this box if you are not using it.
4. Load the micropipette with 20.0μL of green dye by doing the following:
a. Bring the micropipette and the green dye to eye level.
b. Use your thumb to press the plunger to the first stop position, which is your first point of
resistance.
c. Put the pipette tip into the red dye and slowly release the plunger to draw up the solution.
5. Dispense green dye onto the laminated sheet
a. Use your thumb to press the plunger to the first stop position
b. And then press down to the second stop.
c. With the plunger still depressed, pull the pipette out of the tube – this prevents you from
accidentally pulling the liquid back into the tip
6. Take turns in your group practicing with the micropipette.
Homework for Day 1:
Read through the directions for Day 2 again and answer the following questions:
Go to page 5 to answer the following questions:
1.
2.
3.
4.
What are you going to record in this lab packet?
What are you going to scrape with a toothpick?
What are you going to do with the toothpick after you scrape?
What are the two things your group’s materials specialist will do with the tube after transferring
the cheek cells into the tube?
5. What do you have to write in your microfuge tube?
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DAY 2: GETTING THE SAMPLE READY FOR PCR AMPLIFICATION
Materials to collect:
 Toothpick (one for each person in your group)
 One P-20 micropipette for your group
 1.5 mL microfuge tube
 Permanent marker
 Tip box
 Used tip container
Timekeepers, make sure
your group remains on task
1. Materials Specialist, pick up the materials from the table near the window.
2. Wait until you are given a Chelex tube by the teacher. Note that this tube is identified with a
number and letter. Record this number and letter here. Only you will know this anonymous code.
Code:___________ [Rewrite this code on the page with your lab
questions]
3. To collect buccal epithelial cells, use a sterile toothpick to gently
scrape the inside of both cheeks. This procedure should be
noninvasive, so don’t draw blood. (Refer to Figure 2)
4. Transfer the cells that you have removed from the toothpick to the
Chelex tube. Vigorously twirl the toothpick with the Chelex resin to
knock of the cells from the toothpick.
Figure 2: cheek swab
a. This is important: you want to get as many cells off the
toothpick and into the Chelex tube as possible.
5. Close the Chelex tube tightly and give the tube to your materials specialist.
6. The materials specialist will take the tube to the boiling water bath or 100°C
hot block. Boil or heat the cells for 10 minutes. This heating will lyse the cells
and help to destroy some of the nucleases, which degrade the DNA.
a. While the materials specialist is heating your sample, take the microfuge
tubes (refer to Figure 3) and label it with your anonymous code
7. The materials specialist will then take the Chelex tube to the high-speed
centrifuge. Put the Chelex tube inside the centrifuge and let it spin for 5
Figure 3:
minutes. If you are unsure on how to do it the day of, please ask Ms. Judilla for microfuge tube
help.
8. Materials specialist bring back the tubes to your table. Everyone, get the tube with your
anonymous code from the Materials specialist.
9. One by one, use the P-20 micropipette and a clean pipette tip,
carefully remove the 20 μL of supernatant and place it into the
clean 1.5 mL microfuge tube you labeled earlier. Avoid
aspirating Chelex beads as this will inhibit the downstream PCR
procedure.
a. Refer to Figure 4. The supernatant is the gray substance
depicted in the picture that is on top of the pellet.
b. Between each student, dispose the pipette tip into the
Figure 4: supernatant
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used tip container and get a new one from the tip box
10. Give your microfuge tube to the Materials Specialist.
11. Materials Specialist, bring the sample at the front of the room in the rack labeled “Genomic DNA
sample.” Ms. Judilla will perform the PCR amplification and gel electrophoresis .
Homework for Day 2: About PCR
Go to Edline and click on the PCR Virtual Lab link. Read through the page and go through the simulation.
Go to page 5 to answer the following questions
1. What is a primer and why is it necessary in PCR?
2. What is the function of DNA polymerase?
3. What are nucleotides?
4. What is the final product of PCR?
5. What is the purpose of PCR?
6. Briefly explain how PCR works.
DAY 3: ANALYSIS OF GEL ELECTROPHORESIS PHOTO
Overview:
The purpose of gel electrophoresis is used to separate and identify a mixture of biomolecules including
DNA. The components of the mixture can then be identified by their location in the gel. Gel
electrophoresis works based on the fact that the biomolecules have a negative charge, which means that
they will move in response to an electric charge. The
biomolecules move through a gel, and their speed varies
according to their weight, although molecular shape and
degree of charge also influence their movement.
The electrophoresis setup consists of a box containing an
agarose gel and two electrodes that create an electric field
across the gel when the box is attached to a power supply.
Samples of
biomolecules are
pipetted into wells
near the negative electrode. The samples move through the gel
toward the positive electrode.
How to Read the Gel Photo
First column: standard ladder
Second column: homozygous recessive
(Alu-Alu-)
Third column: homozygous dominant
(Alu+Alu+)
Fourth column: heterozygous (Alu+Alu-)
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Day 1 Homework:
1. What are you going to record in this lab packet?
2. What are you going to scrape with a toothpick?
3. What are you going to do with the toothpick after you scrape?
4. What are the two things your group’s materials specialist will do with the tube after
transferring the cheek cells into the tube?
5. What do you have to write in your microfuge tube?
Day 2 Homework
1. What is a primer and why is it necessary in PCR?
2. What is the function of DNA polymerase?
3. What are nucleotides?
4. What is the final product of PCR?
5. What is the purpose of PCR?
6. Briefly explain how PCR works.
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Lab Questions
Code:
Glue the photo of your gel in the space provided below:
1. What is your genotype with respect to the tPA gene? Explain how you determined your
genotype.
2. As a whole class, complete table A:
Table A:
Genotype
Number of Students
+
+
Alu Alu
Alu+AluAlu-AluTotal # of students:
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3. Using the class information gathered in table A, complete table B:
Table B:
Genotypes
Alu+ alleles
Alu+Alu+
Alu- alleles
Alu+AluAlu-AluTotal Alu+ alleles:
Total Alu- alleles:
4. Calculate the frequency (percentage) of each genotype present. Show your work.
5. Calculate the frequency of each allele present in your class. Show your work.
6. The tPA gene that we analyzed codes for a protein given to heart attack patients to help
dissolve blood clots. If the presence of the Alu element (Alu+ element) reduces blood clots, this
would suggest the presence of the Alu element reduces an individual’s risk to suffer from heart
attack. Are your classmates at risk for heart attack? Justify your reasoning with data from the
lab.
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Honors/Extra Credit Assignment - Comparing results from the other class:
a. Calculate the frequency (percentage) of the genotypes present in the other class.
b. Calculate the frequency (percentage) of alleles present in the other class.
c. Is there a difference in relative frequencies of genotypes and alleles? If there is a
difference, explain what the differences are while using evidence from your data.
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