Post-lab 4 Assignment

Due the day of your lab 5A at 6:00 am to the drop box on Avenue.

1) Include a screenshot of your results from the virtual electrophoresis activity (your actual
results and the ideal results must be visible in your screenshot). (1 mark)

2) Why did the bands of DNA in lanes 2, 3, 4, and 5 migrate approximately the same distances
when comparing Student A and Student B? How does this relate to the primer sets and
DNA templates that were used during the PCR reaction? (2 marks)

The bands in lanes 2, 3, 4, and 5 migrated similar distances because they are of similar size.
This relates to the primer sets and DNA templates used during PCR because a certain
primer will cut fragments of similar sizes, thus the same primer likely cut the bands in lanes
2-5.

3) What would need to have been done during the gel electrophoresis procedure in order to
separate all of the 100 bp ladder bands in the Figure 4A-7 and have them appear as
individual bands in the 1.5% agarose gel? (1 mark)

To separate all of the 100 bp ladder bands during gel electrophoresis, it would be necessary
for the bands to remain in the gel for a longer amount of time.

4) Imagine that you repeat the DNA extraction, PCR, and gel electrophoresis experiment using
Unknown Student A and B's DNA. After imaging the resulting gel, only the band
representing the amylase gene fragment is present in Lane 2 of the gel in Figure 4A-7. You
do not observe a band for the actin gene fragment in Lane 2, but you do observe an actin
 band in the samples in Lanes 3, 4, and 5. All four lanes 2-5 have the amylase band. What
could possibly explain why you do not see a band for actin in lane 2? (1 mark)

A potential reason why no band is visualized for the actin gene in lane 2 is due to
experimenter error: While pipetting, if excess bubbles appeared in the well it could result in
a fragment being lost in the gel.

5) What are the diploid amylase (AMY1A) gene copy numbers for Unknown Student A and
Unknown Student B as calculated from your ImageJ and Excel analysis? Include the table
from the excel file that you used to calculate their gene copy number. (2 marks)

Unknown Student A: 7
Unknown Student B: 3

6) Attach the scatter plot figure showing the relationship between gene copy number and
amylase enzyme concentration that you created using the data in Table 4A-3. Follow the
formatting instructions given in this lab manual and in the Excel Tutorial #1 to create a
publication quality figure. Don't forget to include a caption with your graph title, sample
size, and correlation coefficient (r). (5 marks)

The data displayed on the above graph compares AMY1A gene copy number in relation to
salivary amylase concentration in mg/ml. A sample size for the data is a gene copy number of 7
is correlated to an amylase concentration of 0.495 mg/ml. Finally, the correlation coefficient (r)
is equal to 0.57613512.

7) What can you determine from the r value about the relationship between gene copy
number and amylase enzyme concentration? (1 mark)
The correlation value of r=0.57613512 indicates a moderately positive relation between gene
copy number and amylase enzyme concentration.

8) Do Unknown Student A and Unknown Student B’s self-reported starch levels, AMY1A gene
copy numbers, and salivary amylase concentrations support an association between gene
evolution, gene copy number, and amylase enzyme production? Please use Unknown
Student A and Unknown Student B’s data to explain and support your choice for the
presence or absence of an association. (2 marks)

Their starch levels, AMY1A gene copy numbers, and salivary amylase concentrations do not
support an association between gene evolution, gene copy number, and amylase production.
For example, I found that Unknown Student A has a gene copy number of 7, which is the same
as Student K. However, their amylase concentration values differ; 0.495 mg/ml and 1.013
mg/ml, respectively. Additionally, Unknown Student B and Student O have a gene copy number
of 3, but their amylase concentration values are 1.82 mg/ml and 0.43 mg/ml, respectively.

9) Submit a labelled picture (similar to Figure 4A-7) of your restriction digest gel image from
Lab 4B. Number the lanes on the gel and provide a brief description of what is present in
each lane (similar to the descriptions in Table 4A-2) (5 marks).

Lane: 1 2 3 4 5 6

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6

100 bp ladder C H E Bam BgI

10) Submit your correctly formatted 100 bp ladder standard curve that you created in Excel.
Follow the instructions in the lab 4B manual and Excel Tutorial #2 to complete your
publication quality figure. (5 marks)

11) What are the lengths of the DNA fragments, in base pairs, for the two bands that are seen
in all of lanes 2 to 5 of Figure 4A-7? Show your calculations for both bands using the
equation of the line generated from your 100bp standard curve. Which base pair value
represents the amplified DNA that was targeted by the primers that were specific to a
segment of an actin gene? Which base pair value represents the amplified DNA that was
targeted by the primers that were specific to a segment of an amylase (AMY1A) gene? (4
marks)

Unknown Student A: Actin Gene (66mm)

y=27289e-0.068x
y=27289e-0.068(66)
y=27289e-4.488
y=306.81 bp

Unknown Student A: AMY1A Gene (79mm)

y=27289e-0.068x
y=27289e-0.068(79)
y=27289e-5.372
y=126.75 bp

Unknown Student B: Actin Gene (65mm)

y=27289e-0.068x
y=27289e-0.068(65)
y=27289e-4.420
y=328.40 bp

Unknown Student B: AMY1A Gene (77mm)

y=27289e-0.068x
y=27289e-0.068(77)
y=27289e-5.236
y=145.22 bp

Grade: /29