Complete the following tasks.

You discovered that a species of bacteria can break down Styrofoam™ (polystyrene) products due to an
enzyme it produces, polystyrenase. You wish to study the gene that codes for this enzyme.

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: SDS is commonly used in laboratory as component of buffer for cell lysis, cell lysis
during DNA extraction and mostly in SDS-PAGE running buffer. Indeed, SDS is an anionic
detergent applied to protein sample to linearize proteins and to impart a negative charge to
linearized proteins. __________________________________

b. Adding RNase A and Proteinase K during extraction

Answer: RNase A is an endoribonuclease that specifically hydrolyzes RNA 3´ of pyrimidine
residues and cleaves the phosphodiester linkage to the adjacent nucleotide. RNase A is used to
remove RNA during procedures for the isolation of plasmid and genomic DNA. proteinasee K is
used mostly in DNA and RNA extraction protocols. ... To prevent potential digestion of your samples,
proteinase K is inactivated after incubation. The common temperature for inactivation is 95°C. Even in
the typical mouse-tail protocol, proteinase K is regularly used to inhibit harmful
nucleases__________________________________

c. Adding ethanol before recovering the DNA extract

Answer:The main role of monovalent cations and ethanol is to eliminate the solvation shell
that surrounds the DNA, thus allowing the DNA to precipitate in pellet form. Additionally, ethanol
helps to promote DNA aggregation. __________________________________

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.
a. DNA polymerase isolated from Thermus aquaticus
Answer:aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq
polymerase was identified as an enzyme able to withstand the protein-denaturing conditions (high
temperature) required during PCR. __________________________________

b. Deoxynucleotide triphosphates (dNTPs)

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.________________________
 c. Forward and reverse primers
Answer: _________________Two complementary single strands of DNA are released
during denaturation. The forward primer binds to the template DNA, while the reverse primer binds to the
other complementary strand, both of which are amplified in PCR reaction._________________

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).

c. Approximate the size (in kb) of the polystyrenase gene. Write your answer above the band
corresponding to the gene.

Bonus: If you wish to identify the bacterial species in this scenario, what gene is most commonly and
routinely sequenced?
Answer: __________________________________