Lab 9 (Part I) Homework Assignment for Online Students PDF

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BIOL1500 Lab 9 PCR Virtual Lab Worksheet and Homework Questions VIRTUAL LAB WORKSHEETLAB 9 (PART I) HOMEWORK ASSIGNMENT FOR ONLINE STUDENTS Go to https://www.labxchange.org/library/items/lb:LabXchange:f7f6962a:lx_simulation:1. Answer the following questions in the spaces provided. 1. PCR is a lab technique that can be used to generate _______ of copies of specific DNA sequences. Huge numbers

2. What do magnesium ions do during a PCR reaction? What solution are they found in? Magnesium ions are found in the PCR buffer with optimum pH and salt conditions. Magnesium ions serve the purpose of stabilizing the DNA and activating the DNA-synthesizing enzyme Taq polymerase. 3. The region of DNA targeted for amplification is called what? The region of DNA targeted for amplification is called template DNA.

4. The four nitrogenous bases found in DNA are represented by the letters A, T, G and C. What do each of these stand for (i.e. what is the full name of each nitrogenous base) and which ones form complementary pairs? The names of the four nitrogenous bases are adenine, thymine, guanine, and cytosine. Adenine and Thymine form complementary pairs and Guanine and Cytosine form complementary pairs.

5. What kind of chemical bonds are formed between complementary nitrogenous bases? Hydrogen bonds are formed between complementary nitrogenous bases.

6. True or False: primers are required for helicase to unwind the double-stranded DNA template. False

7. Why are dNTPs provided during the PCR reaction? The dNTPs are the building blocks of DNA that are provided in excess during the PCR reaction in order to construct huge numbers of copies of the specific DNA sequences.

BIOL1500 Lab 9 PCR Virtual Lab Worksheet and Homework Questions

PCR

8. What are the five components required to perform PCR? The five components required to perform PCR are a buffer, template DNA, a set of primers, dNTPs,  olymerase. and Taq p

9. After adding the aforementioned components to your PCR tube, what is the name of the machine your sample will be placed in? The sample will be placed in the thermocycler.

10. Explain the three steps of PCR. 1. Denaturation The PCR reaction mixture is heated to 95°C and the hydrogen bonds in DNA are broken. At this step, all of DNA exists as a single strand. 2. Annealing During this step, the temperature of the PCR tube is lowered to allow the primers to bind to their complementary regions on their single stranded DNA templates. 3. Elongation/Extension During this step, the temperature is raised to 72°C for optimal function of Taq p  olymerase. Taq polymerase extends the primers by adding complementary dNTPs in the 5’ to 3’ direction until the end of the DNA template.

11. Define the Tm of DNA. The Tm of DNA is the temperature at which 50% of double-stranded DNA is changed to single-stranded DNA.

12. How many cycles of PCR are required to generate two pairs of new DNA strands of the desired length? Three cycles are required. 13. How do the final quantities of total DNA, desired DNA regions and overhang product copies differ at process efficiencies of 60% vs. 100%? At a 60% efficiency the amount of DNA yielded is only a fraction of what is yielded at 100% efficiency. At 60% efficiency the final quantity of DNA was 237, the desired region was 201, and the overhang product copies was 36. For comparison, the final quantity of DNA at 100% efficiency was 1,073,741,824, the desired region was 1,073,741,764, and the overhang product copies was 60.

BIOL1500 Lab 9 PCR Virtual Lab Worksheet and Homework Questions

14. In PCR, the number of copies of the desired template double every cycle (i.e. the first cycle produces 2 copies, the second produces four copies, the third produces 8 copies, and so forth). Exactly how many copies should be produced in the thirty cycles of PCR that typically occur? After thirty cycles of PCR, there should be 1,073,741,824 copies of desired template DNA. 15. How are the products of PCR visualized? Briefly explain the technique. The products of PCR are visualized using a graph. It is known that PCR doubles the amount of desired template every cycle. The x axis graphed the number of cycles while the y axis graphed the number of copies produced. The graph progressed exponentially with the slope of the graph increasing after every cycle. A logarithmic scale was also used to visualize PCR and showed that the number of copies increased linearly when examined on a logarithmic scale.

HOMEWORK ASSIGNMENT QUESTIONS Answer the following questions in paragraph form. Note: Some questions may require additional research (e.g. a Google search) to complete. 7. You learned in the simulated PCR lab that magnesium ions are important for DNA stabilization and activation of Taq p  olymerase. Explain their relation to the InstaGene matrix used in the lab manual procedure. a. The InstaGene matrix used in the lab contained negatively charged microscopic beads that grabbed the metal ions such as magnesium ions that would have acted as cofactors to enzymes that would degrade DNA. This allowed the DNA to be extracted without degradation. 13. Consider that you are creating a primer for use in the identification of a DNA sample from a crime scene. You are trying to generate a primer that will only bind to a portion of template DNA from the perpetrator’s genome. Would you create a primer that has a high specificity (meaning that it will only bind to a very unique portion of template) or one with a low specificity? Why The best option would be to create a primer that has a high specificity because the primer a. The primer would need to have high specificity since the sequence the primer needs would need to bind to another primer with a very specific sequence....