BIO250 Lab 7 FINAL

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Lab 7 Microbial Genetics & Genetic Engineering BIO250L ” Student Name: Ani Harutyunyan Access Code (located on the lid of your lab kit): AC-Z2CDS9 Lab Report Format Expecta0ons U"lize college level grammar and professional forma4ng when comple"ng this worksheet. Submissions without proper forma4ng, all required photos or suﬃcient responses will be rejected. Pre-lab Ques>ons 1. Why are the concepts in Lab 7 important in the field of microbiology? The techniques of DNA extracIon, cloning, and PCR are crucial in microbiology because they allow for the examinaIon, alteraIon, and increase of geneIc material, playing a crucial role in geneIc studies, biotechnology, and the comprehension of microbial geneIcs. 2. Which DNA nitrogenous bases pair with each other? Adenine&Thymine, Guanine&Cytosine 3. Which bases are purines, and which are pyrimidines? Adenine and guanine=double-ringed purines, cytosine and thymine=single ringed pyrimidines 4. How is DNA informaIon used to make proteins? What are the steps of this process, and what are the significant pieces of cell machinery that parIcipate in this process? The data in the mRNA molecule is decoded into amino acids. 5. What occurs during each of the three steps involved in the PCR cycle? How has the use of PCR changed biotechnology? PCR is used when you have a small amount of unidenIfied DNA isolated from a sample. It's applicable in genotyping, cloning, and forensic analysis. 6. What are the three-nucleoIde sequences that correspond to a specific amino acid called? Codons 7. How could you take a known sequence of amino acids and use it to create an arIficial gene? Hint: Think about this quesIon in the context of QuesIon 6, above. To construct a syntheIc gene based on a known amino acid sequence, it's necessary to determine the codons for each amino acid. The gene is assembled by linking these codons and incorporaIng suitable start and stop codons.

Lab 7 Microbial Genetics & Genetic Engineering BIO250L ” EXPERIMENT 1: EXTRACTION OF DNA Introductory Ques>ons 1. What is the purpose of the following reagents in the experiment? a. Salt (in the DNA extracIon soluIon): neutralizes the DNA's phosphate charge, facilitaIng its precipitaIon, and then permits the DNA to adhere.. b. Detergent (in the DNA extracIon soluIon): Breaks down the membrane, releasing DNA, proteins, and RNA from the cell. c. Ethanol: encourages ionic bonding and extracts the DNA from the soluIon. 2. What do you expect to see when you perform Step 7 in this experiment? In Step 7, the addiIon of ethanol is expected to cause DNA to precipitate, forming visible white strands and air bubbles at the ethanol-fruit soluIon boundary. Data and Observa>ons Insert a photo of your DNA collected on the wooden sIr sIck. Include your name and access code handwri‘en in the background of your photo. The photo must show the DNA as denoted in the lab, using the wooden dowel to remove it from the vial. Results and Discussion 1. Was the DNA soluble in the aqueous soluIon or in the alcohol? aqueous 2. What else could have been present in the ethanol/aqueous interface? How could you have eliminated this? AddiIonal impuriIes from the fruit extract at the ethanol-water boundary can be removed

Lab 7 Microbial Genetics & Genetic Engineering BIO250L ” through extra purificaIon steps, like ethanol washing or a more thorough DNA purificaIon method. 3. What was the texture and consistency of the DNA when you collected it on the wooden sIr sIck? The DNA collected on the wooden sIr sIck most likely had a slimy and stringy texture, forming white strands or clusters that could be coiled or gathered. EXPERIMENT 2: CLONING A DNA FRAGMENT INTO A BACTERIALLY DERIVED PLASMID VECTOR Introductory Ques>ons 1. What is the objecIve of Experiment 2, in simple terms? the objecIve is to insert a foreign gene into a bacterial plasmid using recombinant DNA technology, creaIng a recombinant plasmid that bacteria can replicate, serving various biotechnological purposes. Data and Observa>ons In the table below, list the number of base pairs in the longest length in base pairs for both types of DNA - foreign, and plasmid. Table 1: Fragment Lengths DNA Type Longest Length (in base pairs) Foreign 720 Plasmid 2804

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Lab 7 Microbial Genetics & Genetic Engineering BIO250L ” Results and Discussion 1. What is the expected size of the plasmid plus the cut foreign DNA? 3524 2. What type of ends do the enzymes BamHI and EcoRI produce? How does this type of end facilitate cloning? The BamHI and EcoRI enzymes create double bonds and generate sIcky ends, which aid in the cloning process. DNA ligase can then join the plasmid DNA with the host genomic DNA in the correct orientaIon. 3. What enzyme is necessary to permanently link the digested foreign and plasmid DNA together to form the recombinant DNA molecule? How does this enzyme work? Ligase is the essenIal enzyme that permanently connects the digested foreign and plasmid DNA to create the recombinant DNA molecule. It accomplishes this by uIlizing ATP to create covalent bonds, linking the phosphate sIcky end to the DNA strand sIcky end. 4. How would you clone a gene into a plasmid if there were no common restricIon sites between the two DNA sequences? In cases where there are no shared restricIon sites between the two DNA sequences and the need arises to clone a gene into a plasmid, electroporaIon can be an alternaIve method.

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