PV92 PCR
IMPORTANT: SAVE THIS DOCUMENT WITH YOUR NAME TO YOUR COMPUTER.
Name _____________________
RESULTS
I reran the gel with Ethidium Bromide (EtBr). The picture did not scan well since it was primarily a
black background so I did my best to draw the bands in paint. The initials on 2 tubes were hard to
read. I’m going to guess that J is Josephine and the last one was Henrietta.
The ladder DID NOT work. Another interesting observation is that because EtBr was added I could
observe the bands of DNA moving. The loading dye DID NOT MOVE AHEAD OF THE BANDS.
The loading dye is CRITICAL in letting us know where our DNA is on the gel. I believe we
thought the DNA was not moving, electrophoresed longer and ended up running the DNA off the
gel. Carefully review the interpretation of results on page 59 of the procedure manual (you
may have to go to the online document on our lab page). Study Figures 12 and 13
CAREFULLY. Here are the results I obtained.
If you recall this kit was shipped to our mail room and by the time I got it the frozen reagents had
thawed out. I was assured by the company that this would not affect the results.
INTERPRETATION OF RESULTS
Instructions: Review the results above. Looking at each result as a stand alone result, type ONE of
the following interpretations into the row next to the control or student:
 INVALID,
 homozygous positive PV92
 heterozygous
 homozygous negative PV92
Sample
+/+
+/=
=/=
HL
GC
CL
J
BP
? (Henrietta?)
Interpretation
PV92 PCR
Evaluate Results
Please answer the following questions which are related to evaluation of the results:
1. Which of the controls did not work?
2. What is your explanation for the ladder, and if appropriate, the control(s) not working.
Study Questions
Refer to your procedure manual to answer the following questions:
1. What function does the InstaGene matrix perform?
2. State the 2 functions of the 56C incubation and the function of the 100C incubation step during
the preparation of the DNA sample from cheek cells.
3. State why the InstaGene product not contaminate the DNA sample prior to amplification.
4. State the five components of the master mix and the purpose of each.
5. List the three step cycle, including temperature used, in the polymerase chain reaction and the
purpose of each.
6. Why is it necessary to have a primer on each side of the DNA segment to be amplified?
7. How did Taq DNA polymerase acquire its name?
8. State the two components of the loading dye and the purposes of each in the electrophoresis
reaction.
9. Use Lesson 3 in your procedure manual to answer the following:
a. Define intron.
b. Define exon.
c. What percentage of DNA is “noncoding”?
d. Describe the “Alu sequence” and the clinical significance of this gene.
e. How many base pairs do the primers bracket a sequence in the PV92 region if 1) the
intron does not contain the Alu insertion gene and 2) if Alu is present.
i. No Alu 641 base pairs
ii. Alu present 941 base pairs.
f. Which of the two potential products formed after PCR amplification for Alu will move
the farthest during electrophoresis? 641 – no Alu present.
g. Describe the three distinct outcomes when the PCR products are electrophoresed.
i. No Alu single band of 641 base pairs
ii. Heterozygous for Alu – 2 bands, 941 and 641 base pairs
iii. Homozygous Alu positive – 1 band comprised of 941 base pairs.
10. Describe the process of electrophoresis of DNA fragments.
a. Electrophoresis separates DNA according to their relative sizes.
b. DNA loaded into agarose gel, submerged in conductive buffer.
c. Direct current is passed between wire electrodes at each end.
d. DNA fragments are negatively charged and will move toward the positive pole and
repelled by negative pole.
e. The matrix of agarose gel acts as a molecular sieve through which smaller DNA
fragments can move more easily than larger ones.
f. Smaller fragments will travel farther than larger ones.
g. Fragments of the same size stay together and migrate in what appears as a single band of
DNA on the gel.
11. State the charge of molecules which migrate towards the anode and cathode. What color is each
wire?