MLAB 2337 Molecular Diagnostics Techniques
Laboratory 8: PV92 PCR
INSTRUCTIONS
 In this lab you will perform a DNA extraction, PCR and gel electrophoresis.
 Please review Laboratories 5-7 PRIOR to coming to lab. Answer and submit the study
questions for EACH laboratory into “Assignments” in BlackBoard.
 Review the PV92 Instruction booklet and complete the “Laboratory Prep” section PRIOR to
lab.
 Copy/paste the results into a NEW document. Save the file as “PV92 PCR Results YOUR
NAME”. The study questions will be uploaded SEPARATELY.
RECORDING RESULTS
Instructions: Draw the bands formed on the gel in the appropriate column. Write the initials of
each individual in the column provided.
Ladder
+/+
+/=
=/=
Initials-
Initials-
Initials -
Initials-
Initials -
Initials -
Initials -
INTERPRETATION OF RESULTS
Instructions: Review your results. 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 – no bands or inadequate number of bands (ladder)
 homozygous positive for PV92
 heterozygous positive for PV92
 homozygous negative for PV92
Sample
Ladder
Homozygous positive control
Heterozygous control
Homozygous negative control
Student
Student
Interpretation
EVALUATION OF RESULTS
Please answer the following questions related to evaluation of the results:
1. Did the controls work?
2. Did the ladder give expected results?
3. If “no” to either question, give a possible explanation.
1
Valid? YES or NO
MLAB 2337 Molecular Diagnostics Techniques
PV92 PCR
Laboratory Prep
List the materials, equipment and supplies needed for each of the following including storage
temperature IF important.

DNA Prep

PCR Prep

Gel Prep – Add ethidium bromide to agar prior to pouring gel.
2
Name _________________________________
Date _______________________
MLAB 2337 Molecular Diagnostics Techniques
Laboratory 8: PV92 PCR
Study Questions
Laboratory: PV92
Points:
/36
INSTRUCTIONS FOR STUDY QUESTIONS
1. ADD YOUR NAME AT THE TOP.
2. Save the file as “Lab8PV92 PCR YOUR NAME”
3. Upload to Assignments
Refer to your procedure manual to answer the following questions:
1. What function does the InstaGene matrix perform? WHY is this important? (2 points)
a.
b.
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. (2 points)
a.
b.
3. State why the InstaGene product not contaminate the DNA sample prior to amplification. (1
point)
4. State the five components of the master mix and the purpose of each. (5 points)
a.
b.
c.
d.
e.
5. List the three step cycle, including temperature used, in the polymerase chain reaction and the
purpose of each. (3 points)
a. Cycle 1
b. Cycle 2
c. Cycle 3
6. Why is it necessary to have a primer on each side of the DNA segment to be amplified? (1
point)
7. How did Taq DNA polymerase acquire its name? (1 point)
8. State the two purposes of the loading dye in the electrophoresis reaction. (2 points)
a.
b.
3
9. Use Lesson 3 in your procedure manual to answer the following (7 points):
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.
f. Which of the two potential products formed after PCR amplification for Alu will
move the farthest during electrophoresis?
g. Describe the three distinct outcomes when the PCR products are electrophoresed.
10. Describe the process of electrophoresis of DNA fragments. (7 points)
a.
b.
c.
d.
e.
f.
g.
11. State the charge of molecules which migrate towards the anode and cathode. What color is each
wire? (3 points)
a.
b.
c.
4