Adam Muzzarelli

9/30/17
Stem 1/2
Alu PCR Lab Write Up

Purpose The purpose of this lab was to practice lab procedure and trace where peoples DNA
comes from. We did this using PCR and gel electrophoresis

Hypothesis If my DNA is sequenced using agarose gel electrophoresis and I analyze any Alu
repeats, then it should be possible to find DNA similarities between other people and me and
find ancestors.

Procedure
1. Get a cup containing 10 mL of saline solution (salt water) and swirl vigorously in mouth for 30
seconds

2. Expel the solution back into the cup with the now suspended mouth cells

3. Get a 1.5 mL tube and label it so you can recognize it later

4. Transfer 1500L (1.5mL) of the saline/cell suspension into the labeled tube

5. In a microcentrifuge, spin the tube for a minute to pellet the cells

6. Observe your cell pellet at the bottom of the tube. Show your pellet to your teacher to make
sure it is sufficient. If your pellet is too small, pour off the supernatant and repeat steps 4-5. This
will increase the number of cells in your tube because you will add more saline rinse. If your
pellet is the right size, pour off the supernatant into your cup, being careful NOT to lose your cell
pellet.

7. Check to make sure you can see your cell pellet and that there is about 100L of saline
covering it. You may need to add more saline to get up to about 100L. Rack or flick tube to
mix, which will resuspend the cell and make an evenly mixed solution.

8. Obtain a tube of Chelex from your instructor. Label with your number or initials.

9. Withdraw ALL of your cell suspension from step 7 and add it to the tube containing Chelex.
Flick tube to mix.

10. If your Chelex (with the cell suspension) is in a normal 1.5mL microfuge tube, take your tube
to a heat block station. Slide a cap lock onto the tube lid and place it in the heat block for 10
minutes. Keep track of your tube in the heat block.
11. After heating, gently remove the cap lock and open the tube to release the pressure.
Caution: the tube will be hot! Close and then rack or shake the tube well and place it in a
centrifuge to spin for 1 minute.

12. Obtain another clean 1.5mL microfuge tube and label it with your number. Also write DNA
on this tube.

13. Holding your tube at eye level, use a P200 to withdraw 50L of supernatant from the
Chelex/DNA tube to the newly labeled tube. Be sure NOT to transfer any Chelex beads.

14. Have someone check the DNA tube to be sure that no Chelex beads were transferred into
it. There should be NO Chelex beads present, as they will interfere with the PCR.

15. Place your DNA tube in the class rack. Your teacher will refrigerate your isolated DNA until
you are ready to prepare your PCR amplification.

1. Obtain a tiny PCR tube. Label it with your PIN number, just under the lip. Note: Keep our
PCR tube on ice when setting up the reaction.

2. Pipet 20 L of Master Mix into your PCR tube. 20 L of Master Mix

3. Change your pipet tip and add 20 L of Primer Mix into your PCR tube. 20 L of Primer Mix

4. With a new pipet tip, add 10 L of your extracted DNA into your PCR tube. Note: Make sure
that all the liquids are settled into the bottom of the tube and not on the side of the tube or in the
cap. If not, you can give the tube a quick spin in the centrifuge. Do not pipette up and down; it
introduces error.

5. Setting up the controls: a. Two students will be asked to set up the positive control reactions
(+C) for the class. They will use the positive control DNA provided in the kit. There should be
enough +C PCR sample for one lane on each gel. b. Another two students will set up negative
control reactions for the whole class (C). They will use sterile water. There should be enough
C PCR sample for one lane on each gel.

6. Check the volume of your PCR tube by comparing it to a reference PCR tube with 50 L in it.
It should be near the thermal cycler, set by your teacher. Note: If the volume of your tube does
not match, see your instructor to troubleshoot. You may need to set up the reaction again.

7. Place your reaction into the thermal cycler and record the location of your tube on the grid
provided by your teacher.
8. The cycling protocol for amplification of Alu PV92: 1) 95C hold for 2 minutes 2) 30 cycles of:
94C for 30 seconds 60C for 30 seconds 72C for 2 minutes 3) 72C hold for 10 minutes 4)
4C hold, infinity

1. Retrieve your PCR tube and place it in a balanced configuration in a microcentrifuge. Spin it
briefly (10 seconds) to bring the liquid to the bottom of the reaction tube.
Note: Make sure the centrifuge adapters are in place before putting the tiny PCR tube into the
centrifuge rotor.

2. Add 5 L of loading dye to your PCR tube.

3. Carefully load 15 to 20 L of the DNA/loading dye mixture into a well in your gel. Make sure
you keep track of what sample is being loaded into each well. Note: Avoid poking the pipette tip
through the bottom of the gel or spilling sample over the sides of the well. Use a new tip for
each sample.

4. One student (or the instructor) should load 5-10 L of 100 bp ladder (molecular weight
marker) into one of the wells of each gel.

5. When all samples are loaded, attach the electrodes from the gel box to the power supply.
Have your teacher check your connections and then electrophoresis your samples at 150 Volts
for 2540 minutes.

6. After electrophoresis, the gels will be ready to stain and photograph.

Data/Observations

We took our agarose gel and ran it at 150 V for 20 minutes (electrophoresis), after we
stained the gel using gel red for 72 hours. In our gel, lanes 1a and 2a are loaded with 100 bp
ladder, so we could compare the sequences in our DNA for sizing. The rest of the lanes (wells)
were loaded with 20 l of a 50 l DNA/ 10 l loading dye solution. Lane 2e is the lane I worked
with. Lanes 1g and 2c appear to have worked correctly, while all the other lanes, including mine,
did not work. The two people who had a sequence show up contain the Alu repeat in their DNA.

Lane set up:

1a 1b 1c 1d 1e 1f 1g 1h (1a = ladder)
2a 2b 2c 2d 2e 2f 2g 2h (1b = ladder)

Data Analysis
As my lane appeared to not have worked, no data can be observed as the results were
inconclusive. Since many people had inconclusive data, our class used a fake set of data to find
the frequency of the Alu repeats represented by the classroom. To do this we used various
equations to find the frequency of (+/+) Alu repeat students, meaning they have alu inserts in
both chromosomes, (+/-) they have the alu insert in just one chromosome, and (-/-) the students
who have no alu inserts.
We then used another equation to figure out the genotype frequency of the different
genotypes in our classrooms population. The hypothesis is correct, if my DNA sequence
actually worked, then I would have been able to find similarities between what Alu alleles I have
and what alleles others have. People with identical alleles would have a common ancestor with
me, since we both have the same Alu repeats. Some of the errors that most likely occurred were
improper measurements, which could lead to the PCR reaction not working. Another error would
be not inserting the dye into the well, which would break the electrophoresis process since the
DNA would disperse into the liquid. One way to improve the lab would be to have the teacher
insert the dyes into the loading wells. This would help improve reliability in the actually dye and
DNA making it to the correct spot so the electrophoresis of the gel will consistently work. This
lab could lead to the further investigation of how each allele came to be, or to find people with
matching genotypes and try to find a common ancestor.

Conclusion
 Performing PCR amplification of DNA is very difficult. This experiment involved taking
DNA from our cheek cells and using PCR to replicate it. We then took the DNA and ran it
through agarose gel electrophoresis, with and end goal of trying to find Alu repeats in our DNA.
PCR is a process which involves various components, DNA, primers, a buffer, DNA polymerase,
and deoxynucleoside triphosphates; it replicates DNA by breaking the double helix into single
strands, and then copying back the second strands and repeating the process. This process can
be very challenging because any mistake with incorrect measurements will mess up the entirety
of the rest of the lab. PCR is an operation which is essential to any lab revolving around DNA.