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Collin McGarty

10-5-17
PCR Lab Report
Purpose: The purpose of this lab was to take DNA from our mouths replicate DNA
segments by using PCR. Then, we wanted to sequence our DNA by using gel
electrophoresis. By doing this, we hoped to learn more about where our DNA came
from. Commented [1]: More concise

Hypothesis: If we successfully followed the procedure and the steps correctly, then we
will have replicated and sequenced DNA segments that can tell us where I came from
and if other people came from similar backgrounds. Commented [2]: What is your expected result?

Procedure: The procedure goes as following, Commented [3]: Cite the packet next time

DNA Preparation Using a Saline Mouthwash


1. Vigorously swirl 10mL of saline solution in your mouth for 30 seconds.
2. Expel saline into a cup and swirl to mix the cells.
3. Label a 1.5mL microfuge tube with a number (like a PIN) or your initials.
4. Transfer 1500L (1.5mL) of the saline/cell suspension into the labeled microfuge
tube. To do this, transfer 750L twice. 1.5 mL saline
5. In a microcentrifuge, spin your saline cell suspension for 1 minute to pellet the cells.
Be sure to use another students sample as a balance.
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. Heat block version: 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.
PCR tube version: If your Chelex (with your cell suspension) is in a tiny PCR tube,
follow your teachers instruction on placing it in a thermal cycler at 99C for 10 minutes.
Record the location of your tube.
11. After heating, gently remove the cap lock and open the tube to release the pressure.
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 P-200 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.
Polymerase Chain Reaction
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.
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.
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 Thermal cycler Instrument displaying program
parameters

Electrophoresis of Amplified DNA


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.
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.
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 electrophorese your
samples at 150 Volts for 2540 minutes.
6. After electrophoresis, the gels will be ready to stain and photograph.

Analysis: The data was inconclusive for my experiment, so I cannot analyze the data.
This same thing happened to many other students in our class, so we made a fake set
for the class to analyze and try to find alu repeats within the set. We then found the
frequencies of different alleles (+/+, +/-, -/-) within the class. If you have a +/+ that
means that you have an alu repeat. If you have a +/-, then you have an alu insert in
only one chromosome and -/- means that you have no alu inserts.
After finding the results with the class set, I found my hypothesis to be correct. If Commented [4]: if you got no data how can you say
correct?
I was to get a correct reading of my DNA and my alleles, I would have been able to
trace my background and compare that with what others found.
Since this was our first experiment, I think that there were some issues with the
lab that could be fixed. First off, we probably didnt get the correct measurements and
amounts that we needed to get. Also, some of the loading dye didnt go into the wells
but instead went into the gel which may have messed up the electrophoresis process. Commented [5]: are these the errors? any more?
We could improve this lab by having a way to measure exactly how much we were
putting in or taking out of the liquids. Another way we could improve this lab would be
to have someone put the loading dye into the wells for us. This would help us because
it would be more dependable than students putting in the loading dye. Commented [6]: Improvements have to be procedural.

Conclusion: In conclusion, we werent able to successfully find our own alu


sequencing, but after using a fake set of data, I was able to prove my hypothesis. In Commented [7]: you cannot prove off of fake data
this experiment, we first got our DNA samples and replicated them using PCR. Then,
we used gel electrophoresis to try to find our sequencing. If things had gone to plan, we
wouldve been able to see where our DNA came from and would be able to trace where
we came from using our data gathered from the electrophoresis. We used the class set
to find alu inserts and where they appear. Then, we would be able to trace our
background using the alu inserts that we found. Commented [8]: Conclusion has to be in CLEAR
format, not a summary of what you did but a summary
of what you learned