Review for the Final Exam-Fall 21 PDF

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Review for the Final Exam-Fall 21 2021/2022/2020/ Review for the Final Exam-Fall...

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Review for the Final Exam Experiment 6: ELISA 1. Describe the basic process of an ELISA. What binds to what? When you get a positive or negative result from an ELISA, what are you actually seeing? 2. What are negative and positive controls? And what are their functions? 3. Given wells containing tested samples, be able to classify them as positive or negative results. 4. Be able to recognize erroneous results and determine potential source(s) of error. Experiment 7: PCR Reaction of Mitochondrial DNA 1. Understand the purpose of a PCR reaction 2. Understand the mechanism of a PCR reaction a) List essential components of a PCR reaction b) What kind of DNA can be used as template? Does it have to be pure? c) The basic properties of PCR primers d) Why should there be primers in the reactions? e) What is special about the polymerizing enzyme used in the reaction? f) What are the three basic steps in each cycle? The temperature used and the purpose of each step g) Is the amplification linear or exponential? h) What determines the size of a PCR product? i) What are the two functions of the dNTPs? j) Why is MgCl2 included in the reaction? 3. Understand how you set up your PCR reaction in the lab.  Isolation of total DNA from epithelial cells (the function of Chelex and boiling) 4. A student performed a PCR reaction and he wants to know if he has obtained the expected PCR product. What does he need to do? 5. What is the basis of separation of DNA on an agarose gel? How is it different from the protein agarose gel (E11) in terms of the setup or the property of the gel? 6. What gives DNA the negative charge? 7. Understand how your PCR product was visualized a) What affect the migration rate of DNAs in agarose gel electrophoresis? b) What chemical “stains” the DNA in our experiment? And how? c) What is the purpose of including a marker lane in the gel? d) How does the size of linear DNA molecules relate to their migration rate? e) What two blue dyes are included in the marker? Why? ( page 3 in E5 procedure) Experiment 8: Bacterial Transformation 1. Define genetic transformation. 2. Compare constitutive vs. inducible gene expression. 3. What are some important characteristics of the strain and the plasmid in this experiment? 4. Understand how bacterial transformation is set up in this lab. a) Why do we use LB/amp media to select for transformants? b) What was the purpose of the arabinose? c) Which plate, LB/amp or LB/amp/arabinose, produced glowing colonies? WHY?

d) What are the control plates? What are their purposes? 5. Be able to determine the transformation efficiency given the number of transformants, the concentration and the volume of plasmid used. Sample question: A single colony of bacteria HB101 was mixed with 800 μl of CaCl2 transformation solution and split into two tubes (+pGLO and –pGLO). 20 μl of plasmid pGLO DNA with concentration of 0.03 μg/μl was added to +pGLO tube. After heat shock, 180 μl LB broth was added to each tube. 150 μl of the cell suspension from (+) pGLO tube was spread onto a LB/Amp plate, and 300 transformants were observed on that plate after incubation. Transformation efficiency = transformants/μg of plasmid DNA Total amount of pGLO DNA used = __________

Fraction of DNA used =________________

Micrograms of DNA spread on the plates =____________

Transformation efficiency =________________ Answer: 0.6 μg, 0.25, 0.15 μg, 2000 transformants/ μg

Experiment 9: Spectrophotometry 1. Know how to prepare a diluted solution from a stock solution using parallel dilution method. 2. Know what an absorption spectrum is AND how it is generated. Be able to graph the data into a properly labeled curve. (title, label, units of measurements, linear scale). 3. Understand the concept of lmax of a chemical and how it is determined experimentally. 4. The following questions are all related to the standard curve showing the relationship between absorbance and concentration: a. What is the purpose of the standard curve? b. Why such a curve is typically measured at the lmax of a chemical? c. Be able to describe the Beer’s law and relate it to the standard curve d. Be able to manually draw and properly label a standard curve provided with data e. Be able to use the standard curve equation generated by Excel to calculate the concentration of an unknown solution. 5. Be able to calculate molar absorptivity e using Beer’s Law. 6. Be able to turn on the visible light and autozero the spectrophotometer, setting a specific wavelength and then accurately measure the absorbance of a solution at that wavelength.

Experiment 10: Wheat Germ Acid Phosphatase Assay 1. What may influence the activity of an enzyme? List at least 4 factors. 2. Describe how the amount of enzyme affects the rate of a reaction. 3. Describe how the amount of substrate affects the rate of a reaction. 4. Understand why reaction rates decline with time and use this information to correctly process the data (by choosing the proper data points to do linear regression) 5. Understand how to construct and read the following two graphs:  The standard curve of absorbance vs nmole/ml of nitrophenol  The amount of nitrophenol produced in the assay vs time a) What is the purpose of each graph? b) How is each graph produced experimentally? c) Be able to determine the Vo given the appropriate graph and equation. d) Be able to critique a sample student graph for any errors 6. Be able to describe the reaction catalyzed by acid phosphatase and describe how the assay was performed, and the functions of KOH in the assay. Experiment 11: Protein Separation by Agarose Gel Electrophoresis (knowledge regarding gel electrophoresis from E5 will be included in exam) 1. Understand the subunits of proteins: amino acids  Be able to label amino group, carboxyl group and side group  Provided with the structure of amino acids, be able to classify them into nonpolar, polar or charged amino acids  Know which two amino acids are acidic amino acids, which three amino acids are basic amino acids, under what condition?  Any amino acid can be charged under appropriate condition. True or False? 2. Define the four levels of protein structures. 3. Understand the concept of pI  The definition  Know the relationship between acidity of amino acid or proteins and pI  Be able to determine the charge of an amino acid or a protein given its pI and environmental pH ; then predict its migration direction in an agarose gel 4. Understand the difference between normal hemoglobin and mutated hemoglobin in sickle cell patients; be able to compare the migration rates of the two types of hemoglobin (HbA and HbS) based on the nature of the mutation. 5. Understand the separation principle of protein agarose gels (native or non-reducing gel electrophoresis)  What determines the direction of movement of the proteins?  What determines the rate of movement of the proteins?  Does the molecular weight of a protein affect its migration?  Does the separation require proteins to be denatured?  Does the protein change its shape while it is moving in the gel?

6. Properly load a sample into an agarose gel. Assemble the gel apparatus and connect to the power supply correctly (the wells would be set at the end of the gel)....