Question 1

Which of the following best describes the role of dNTPs in a PCR reaction?

Accepted Answer

They serve as the building blocks for new DNA strands.

Question 2

You are designing a PCR experiment to amplify a gene region with 20% GC content. What is the most likely challenge you will face?

Accepted Answer

The region will denature too easily, leading to non-specific amplification.

Question 3

If you use dNMPs instead of dNTPs in a PCR reaction, what is the expected outcome?

Accepted Answer

No DNA synthesis will occur because dNMPs lack the necessary triphosphate group.

Question 4

A DNA sequence is 5'-ATGTAG-3'. If you want to clone this into a vector, which of the following is the correct complementary strand?

Accepted Answer

3'-TACATC-5'

Question 5

You are expressing a protein in E. coli and the gene is highly glycosylated. What is the most likely outcome?

Accepted Answer

The protein will lack glycosylation and may be non-functional.

Question 6

Which of the following gene sequences ensures that your expression system contains the desired vector?

Accepted Answer

Antibiotic resistance gene.

Question 7

In chromatography, what is the main function of the stationary phase?

Accepted Answer

To separate components based on their interactions with the matrix.

Question 8

You need to separate a protein with a molecular weight of 70.5 kDa from another protein with 45 kDa. Which chromatographic method is most suitable?

Accepted Answer

Size exclusion chromatography.

Question 9

A protein sample is loaded onto a Ni/Co affinity column. Which buffer pH is most suitable for eluting the protein?

Accepted Answer

$pH = 5.5$

Question 10

During SDS-PAGE, why are SDS and BME added to the sample buffer?

Accepted Answer

To denature proteins and break disulfide bonds for linearization.

Question 11

A protein mixture contains three proteins: 35 kDa, 45 kDa, and 70 kDa. After SDS-PAGE, where will the largest protein appear on the gel?

Accepted Answer

At the top of the gel, closest to the well.

Question 12

You are analyzing a protein sample using size exclusion chromatography. Which protein will elute first?

Accepted Answer

The largest protein.

Question 13

A protein sample is mixed with an impurity. How can you detect the impurity using SDS-PAGE?

Accepted Answer

By observing an extra band at a different molecular weight.

Question 14

Which amino acid absorbs light at 280 nm most strongly?

Accepted Answer

Tryptophan

Question 15

A substrate absorbs light at 320 nm, but when converted to product by an enzyme, it absorbs at 480 nm. What does this indicate about the reaction?

Accepted Answer

The product has a different electronic structure than the substrate.

Question 16

Which of the following is a critical reason for using a regeneration buffer at $pH = 5.5$ in Ni/Co affinity chromatography?

Accepted Answer

It disrupts the interaction between the His-tag and the metal ion, allowing elution.

Question 17

Which of the following best describes a CD spectrum for a protein with predominantly alpha-helix structure?

Accepted Answer

Negative peaks at 208 nm and 222 nm, and a positive peak near 190 nm.

Question 18

You are using ion exchange chromatography to purify a protein. The buffer pH is 5.6. What type of ion exchange should you use for a protein with a pI of 7.3?

Accepted Answer

Cation exchange chromatography.

Question 19

A protein forms a disulfide-bridged dimer with a molecular weight of 70 kDa. After SDS-PAGE with BME, what will you observe?

Accepted Answer

A band at 35 kDa due to reduction of the dimer.

PCR, Cloning, Chromatography, and Protein Analysis: Study Notes

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