microbial-genetics-lab-13

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Studocu is not sponsored or endorsed by any college or university Microbial Genetics - Lab 13 Microbiological Laboratory (University of Southern Maine) Studocu is not sponsored or endorsed by any college or university Microbial Genetics - Lab 13 Microbiological Laboratory (University of Southern Maine) Downloaded by Brenda Ledbetter (bledbetter0627@gmail.com) lOMoARcPSD|28593260

PRE-LAB QUESTIONS 1. Which DNA nitrogenous bases pair with each other? Which bases are purines, and which are pyrimidines?  DNA nitrogenous bases that pair together are Adenine (A) and Guanine (G), (AG) both being purines. Cytosine (C) with Thymine (T), (AT) are pyrimidines. 1. How is DNA information used to make proteins? What are the steps of this process?  DNA information is used to make proteins through the process of transcription and translation. The first step is when the enzymes from the DNA molecules are transcribed into mRNA with help from the RNA polymerase. Next the mRNA reaches the ribosome, tRNA and translated into amino acids that block the proteins. 2. Give an example of a scenario in which you would perform PCR vs a scenario in which you would use recombinant DNA technology.  An example of performing PCR would be when determining the source of an illness and could also be used to get DNA from blood samples. An example of using recombinant DNA technology could be when using human growth hormones. 3. What occurs during each of the three steps involved in the PCR cycle? How has the use of PCR changed biotechnology? ©eScience Labs, 2018 Microbial Genetics and Genetic Engineering Downloaded by Brenda Ledbetter (bledbetter0627@gmail.com) lOMoARcPSD|28593260

 The three steps involved in the PCR cycle are denaturing, annealing, and elongation/extending. Denaturing is when the DNA is heated and separated into two single strands. Annealing happens next and this is when the temperature cools down and the DNA primers attach to the DNA template. The final step is elongation/extending the temperature goes back up and the new strand of DNA is made. The use of PCR changed biotechnology because it can be used to identify pathogens and used in medical applications. 4. How could you take a protein with a known sequence of amino acids and use it to create an artificial gene?  Having protein with a known sequence of amino acids can be used to create an artificial gene. This can be done by using recombinant DNA technology such as bioinformatic tools and databases. ©eScience Labs, 2018 Microbial Genetics and Genetic Engineering Downloaded by Brenda Ledbetter (bledbetter0627@gmail.com) lOMoARcPSD|28593260

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EXPERIMENT 1: DNA EXTRACTION Post-Lab Questions 1. What is the purpose of the following reagents in the experiment? a. Salt (in the DNA extraction solution): The purpose of salt is used to neutralize the charge of the DNA’s phosphate, enable the precipitation, and allows the DNA to attach. b. Detergent (in the DNA extraction solution): The purpose of Detergent is used to break the membranes and release the DNA, RNA, and proteins from the cells. c. Ethanol: The purpose of the Ethanol is used to promote ionic bonds and cause precipitation, which takes the DNA out of the solution. 2. What else might be in the ethanol/aqueous interface? How could you eliminate this?  RNA and soluble salt might be in the ethanol/aqueous interface. This could be eliminated by using the centrifuge, RNase enzymes, or crushing in a bag and draining through a cheese cloth. 3. What is the texture and consistency of the DNA?  The texture and consistency of the DNA is fragile, sticky, and slimy. 4. Is the DNA soluble in the aqueous solution or in the alcohol? ©eScience Labs, 2018 Microbial Genetics and Genetic Engineering Downloaded by Brenda Ledbetter (bledbetter0627@gmail.com) lOMoARcPSD|28593260

 The DNA is soluble in the aqueous solution. ©eScience Labs, 2018 Microbial Genetics and Genetic Engineering Downloaded by Brenda Ledbetter (bledbetter0627@gmail.com) lOMoARcPSD|28593260

EXPERIMENT 2: CLONING A DNA FRAGMENT TO A BACTERIALLY-DERIVED PLASMID VECTOR Data Tables Table 1: Fragment Lengths DNA Type Longest Length (in base pairs) Foreign 720 Plasmid 2804 Post-Lab Questions 1. What is the expected size of the plasmid plus the cut foreign DNA?  The expected size of the plasmid plus the cut of foreign DNA was 3524 base pairs. 2. What type of ends do the enzymes BamHI and EcoRI produce? How does this type of end facilitate cloning?  The enzymes BamHI and EcoRI form double bonds and produce sticky ends that facilitate the cloning process. The plasmid DNA can be linked by DNA ligase in the correct orientation in the host genomic DNA. 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 enzyme that is necessary to permanently link the digested foreign and plasmid DNA together to form the recombinant DNA molecule. This works because ©eScience Labs, 2018 Microbial Genetics and Genetic Engineering Downloaded by Brenda Ledbetter (bledbetter0627@gmail.com) lOMoARcPSD|28593260

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the enzyme uses ATP to form covalent bonds and links the phosphate sticky end to the DNA strand sticky end. 4. How would you clone a gene into a plasmid if there were no common restriction sites between the two DNA sequences?  If there were no common restriction sites between the two DNA sequences and a need to clone a gene into a plasmid, it could be done by using electroporation. ©eScience Labs, 2018 Microbial Genetics and Genetic Engineering Downloaded by Brenda Ledbetter (bledbetter0627@gmail.com) lOMoARcPSD|28593260

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