Post-lab 4 Assignment

Due two days following Lab 4 at 1:00 pm 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 distance that a DNA band travels during analysis is mainly determined by the size of
the DNA fragments. If the bands in lanes 2, 3, 4, and 5 of the gel electrophoresis are
approximately at the same position, it indicates that the DNA fragment sizes are very
similar or identical. This implies that the DNA templates and primer sets used by both
Student A and Student B in their PCR reactions were the same. If they had used different
 templates or primers, the DNA fragment sizes would have varied, resulting in bands that
appeared at different distances on the gel.

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 4-7 and have them appear as
individual bands in the 1.5% agarose gel? (1 mark)

To separate the individual bands of a 100bp ladder and make them clearer, you should run the
gel electrophoresis for a longer time. This allows the DNA fragments to separate more distinctly
based on their sizes. Running the gel until the last band is almost falling off gives the best
results, as it lets the fragments separate for a longer period without losing any bands. Adjusting
the voltage can also help by pulling the DNA fragments faster towards the bottom of the gel,
making the bands more separate and easy to see.

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 4-7. 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)

If the experiment is repeated without any changes to the procedure, we would expect similar
results to the first trial. Therefore, if we don't see the actin band in Lane 2, it suggests that
something went wrong during the procedure. One possible mistake could have occurred while
pipetting. For instance, if the pipette was accidentally pressed too far when adding the sample
to Lane 2, it may have created bubbles in the gel. These bubbles could have disturbed the
sample and caused the actin DNA fragments to be lost.

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)
Student Amylase Area Actin Area Normalized Amylase: Number of
Results Results amylase data Actin Ratio Diploid
Amylase
Gene Copies
Unknown 7211.803 6256.76 20864.68 3.3353 3
Student A
Unknown 11270.137 5065.52 32605.97 6.437 6
Student B

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 4-3. Follow the
formatting instructions given in the lab 4 manual and in 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)

Scatter plot showing the correlation between amylase concentration (mg/mL) and AMY1A gene
copy number in 21 unknown students. The correlation coefficient of 0.663821 indicates a
moderate positive relationship between these variables.

7) What can you determine from the r value about the relationship between gene copy
number and amylase enzyme concentration? (1 mark)
Based on the scatter plot analysis, we can conclude that there is a moderate correlation
between the number of AMY1A genes and the concentration of the Amylase enzyme. This
conclusion is based on the calculated correlation value (r value) of 0.663821, which falls within
the range of 0.5 to 0.8. A moderate correlation implies that the number of AMY1A genes has a
moderate impact on the level of amylase enzyme in an individual.

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)
The data suggests that there is a connection between gene evolution, the number of AMY1A
gene copies, and the production of salivary amylase in Student A and Student B. Student A has
3 copies of the AMY1A gene, while Student B has 6 copies. This means that Student B has more
gene copies, which allows them to produce more salivary amylase (about 0.591 mg/mL), while
Student A produces less (only about 1.947 mg/mL). From this information, we can infer that
Student B's ancestors likely had a diet high in starch. This would explain the higher number of
AMY1A gene copies and the higher concentration of salivary amylase in Student B. On the other
hand, Student A's ancestors probably had a diet with less starch, which aligns with their lower
concentrations of salivary amylase and lower number of AMY1A gene copies. In conclusion, it
can be said that individuals with a high starch diet in their ancestry tend to have a greater
number of AMY1A gene copies, leading to higher production/concentration of salivary amylase.

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

Yellow: unknown student A actin gene(300 bp)

10) 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 4-7? 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)

Trendline Equation: y=33917e^-0.086x

Unknown Student A: Amylase (AMY1A) Gene (67 mm)

Unknown Student B: Actin Gene (56 mm)

Unknown Student B: Amylase (AMY1A) Gene (65 mm)

Grade: /24