BCH5413 Final Exam A+ Questions and
Answers Verified Solutions Latest Update
2026/2027
Question:
Why do you need a loading control for a western blot? What would be an ideal Loading control for
a western blot experiment?
Answer:
Loading control needed to ensure equal loading of sample in all lanes.
Ideal controls for western blotting are housekeeping proteins such as actin or GAPDH, proteins
whose expression should not change with the hormone treatment.
Question:
In a western blot, how would you analyze the quality of the protein sample?
Answer:
Using an example gel and staining it with Coomassie dye to look at the integrity of the samples. If
nice, discrete bands are observed, protein sample is of good quality. Also helps to observe if
loading was even between samples/lanes.
This type of staining can also be done to the membrane itself.
Question:
In a PCR reaction, why do you need:
Both a forward and a reverse primer?
Answer:
The purpose of PCR primers is to provide a "free" 3'-OH group to which the DNA
polymerase can add dNTPs
Question:
In a PCR reaction, why do you need:
Template?
,Answer:
The template DNA is required so that more copies of that DNA can be made
Question:
In a PCR reaction, why do you need:
dNTP?
Answer:
The purpose of the deoxynucleotide triphosphates (dNTPs) is to supply the "bricks."
Since the idea behind PCR is to synthesize a virtually unlimited amount
of a specific stretch of double-stranded DNA, the individual DNA bases
must be supplied to the polymerase enzyme.
Question:
In a PCR reaction, why do you need:
Magnesium?
Answer:
Magnesium is required as a co-factor for thermostable DNA polymerase. Excessive magnesium
concentrations also stabilize double stranded DNA and prevent complete denaturation of the DNA
during PCR reducing the product yield.
Question:
In a PCR reaction, why do you need:
DNA polymerase?
Answer:
PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands
as templates.
Question:
After one cycle of PCR, how many DNA fragments to you have that exactly match your target
sequence? Explain your answer.
Answer:
, When PCR is started with one dsDNA fragment, there will be two exact copies of the target
sequence after one cycle.
For each cycle, the PCR doubles the existing number of copies of DNA sequence = logarithmic
amplification.
Question:
What value was there in the discovery and then use of Thermus Aquaticus DNA polymerase in
PCR? Why did it make the process more efficient, describe specific steps in the process?
Answer:
Thermus Aquaticus DNA polymerase is heat stable.
When PCR was done before this discovery, more polymerases had to be added with each cycle.
The discovery of this thermostable polymerase allowed for automation of PCR method.
Question:
In genotype analysis of a mouse knockout experiment, describe how the two different forward
primers with one reverse primer would allow you to distinguish between a mouse that is
heterozygous, homozygous normal (no knockout), and homozygous knockout.
Answer:
PCR can be used to identify if a mouse is a KO or not.
How a KO mouse is generated: using a targeting vector.
Take a sequence (in Dr. Gumz experiment, she used PGK-Neo) and use it to replace a part of the
gene that you want to KO (Dr. Gumz KO exons 10, 11, and 12 from the Per1 gene in mouse kidney
cells).
To identify if a mouse is heterozygous, homozygous normal (WT), or homozygous KO, three
primers are used:
Two forward primers:
WT forward primer anneals to the normal gene (Dr. Gumz used a forward primer that anneals to
exon 10).
KO forward primer anneals to the sequence that has been KO (Dr. Gumz used a forward primer
that anneals to endogenous sequence PGK-Neo).
One reverse primer that anneals to a portion of exon 13, which should be present in all three
genotypes.
Question:
What is the main principle behind bisulfite PCR?
Answers Verified Solutions Latest Update
2026/2027
Question:
Why do you need a loading control for a western blot? What would be an ideal Loading control for
a western blot experiment?
Answer:
Loading control needed to ensure equal loading of sample in all lanes.
Ideal controls for western blotting are housekeeping proteins such as actin or GAPDH, proteins
whose expression should not change with the hormone treatment.
Question:
In a western blot, how would you analyze the quality of the protein sample?
Answer:
Using an example gel and staining it with Coomassie dye to look at the integrity of the samples. If
nice, discrete bands are observed, protein sample is of good quality. Also helps to observe if
loading was even between samples/lanes.
This type of staining can also be done to the membrane itself.
Question:
In a PCR reaction, why do you need:
Both a forward and a reverse primer?
Answer:
The purpose of PCR primers is to provide a "free" 3'-OH group to which the DNA
polymerase can add dNTPs
Question:
In a PCR reaction, why do you need:
Template?
,Answer:
The template DNA is required so that more copies of that DNA can be made
Question:
In a PCR reaction, why do you need:
dNTP?
Answer:
The purpose of the deoxynucleotide triphosphates (dNTPs) is to supply the "bricks."
Since the idea behind PCR is to synthesize a virtually unlimited amount
of a specific stretch of double-stranded DNA, the individual DNA bases
must be supplied to the polymerase enzyme.
Question:
In a PCR reaction, why do you need:
Magnesium?
Answer:
Magnesium is required as a co-factor for thermostable DNA polymerase. Excessive magnesium
concentrations also stabilize double stranded DNA and prevent complete denaturation of the DNA
during PCR reducing the product yield.
Question:
In a PCR reaction, why do you need:
DNA polymerase?
Answer:
PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands
as templates.
Question:
After one cycle of PCR, how many DNA fragments to you have that exactly match your target
sequence? Explain your answer.
Answer:
, When PCR is started with one dsDNA fragment, there will be two exact copies of the target
sequence after one cycle.
For each cycle, the PCR doubles the existing number of copies of DNA sequence = logarithmic
amplification.
Question:
What value was there in the discovery and then use of Thermus Aquaticus DNA polymerase in
PCR? Why did it make the process more efficient, describe specific steps in the process?
Answer:
Thermus Aquaticus DNA polymerase is heat stable.
When PCR was done before this discovery, more polymerases had to be added with each cycle.
The discovery of this thermostable polymerase allowed for automation of PCR method.
Question:
In genotype analysis of a mouse knockout experiment, describe how the two different forward
primers with one reverse primer would allow you to distinguish between a mouse that is
heterozygous, homozygous normal (no knockout), and homozygous knockout.
Answer:
PCR can be used to identify if a mouse is a KO or not.
How a KO mouse is generated: using a targeting vector.
Take a sequence (in Dr. Gumz experiment, she used PGK-Neo) and use it to replace a part of the
gene that you want to KO (Dr. Gumz KO exons 10, 11, and 12 from the Per1 gene in mouse kidney
cells).
To identify if a mouse is heterozygous, homozygous normal (WT), or homozygous KO, three
primers are used:
Two forward primers:
WT forward primer anneals to the normal gene (Dr. Gumz used a forward primer that anneals to
exon 10).
KO forward primer anneals to the sequence that has been KO (Dr. Gumz used a forward primer
that anneals to endogenous sequence PGK-Neo).
One reverse primer that anneals to a portion of exon 13, which should be present in all three
genotypes.
Question:
What is the main principle behind bisulfite PCR?
