Last Name 1
Student’s Name
Professor’s Name
Course Number
Date
Gene of Interest
Task 1: DNA Extraction
To begin work on the bacterium, you begin by extracting its genomic DNA (gDNA). What is the
purpose of the following procedures? Answer briefly but completely.
a. Using sodium dodecyl sulfate, a detergent
Answer: The purpose of using Sodium dodecyl sulfate (SDS) is to disrupt the nuclear enclosure
and cell membrane to release the chromosomes. The chromosome contains the cell DNA, thus, it
needs to be exposed. After the chromosome is exposed. SDS breaks down the DNA walls such
as histones, exposing the DNA. The SDS, a detergent, has the ability to denature proteins.
b. Adding RNase A and Proteinase K during extraction
Answer: The process of extracting DNA is usually marred with many contaminating proteins
that need to be removed to maintain the purity required. Adding a wide-ranging serine protease
to avert the contamination is necessary. RNase A is used in breaking down contaminants such as
RNA, whereas Proteinase K is for breaking down damaging proteins ensuring they are removed.
These two have been applied in numerous DNA extraction procedures.
c. Adding ethanol before recovering the DNA extract
Answer: The addition of ethanol removes the solvation shell surrounding the DNA by lowering
the dielectric constant, thus, precipitating the DNA like a capsule. In addition, ethanol provides
an environment where the DNA can aggregate. Usually, a 70% solution of ethanol is employed.
Task 2: Polymerase Chain Reaction
After purifying the gDNA extract, you want to isolate and amplify the polystyrenase gene. You
perform PCR using the appropriate gene-targeted primers. What is the purpose of the following
PCR components? Answer briefly but completely.
Last Name 2
a. DNA polymerase isolated from Thermus aquaticus
Answer: The bacterium Thermus aquaticus is tolerant to heat, therefore, it maintains its stability
at extremely high temperatures, that is, it endures protein denaturing. The stability aspect makes
it ideal in the PCR process of thermos cycling.
b. Deoxynucleotide triphosphates (dNTPs)
Answer: Deoxynucleotide triphosphates (dNTPs) in the PC process acts as an elementary unit
forthe strands of the new DNA.
c. Forward and reverse primers
Answer: The reverse and forward primers are designed in a way that flanks the target area for
amplification of the wanted gene. The complementary strand is bound by the reverse
primer,whereas the template DNA is bound by the forward primer.
Task 3: Agarose Gel Electrophoresis
Your amplicon from PCR was subjected to AGE for analysis. You are shown the image of the
gel loaded with the following lanes: (A) negative control, (B) size ladder (1, 2, 3, 4, 6, and 10
kb), (C) gDNA extract, (D) PCR amplicon. However, due to mishaps while loading the gel with
the samples, you are not sure which lane is which. You are shown a diagram of the obtained gel
below.
a. Label each lane of the gel. Write only the corresponding letters in the wells above.
b. Above each band in the size ladder, write its size (in kb).
Last Name 3
Approximate the size (in kb) of the polystyrenase gene. Write your answer above the band
corresponding to the gene
The gene lies between 4kb and 6kb, which gives it an approximate size of 5kb.
Bonus: If you wish to identify the bacterial species in this scenario, what gene is most commonly
and routinely sequenced?
Answer: 16s ribosomal RNA gene is routinely and commonly used to identify the bacteria.
