BIOL&160 lab, Spring 2020                                                    Name: ____________________________________

DUE: June 8-12, 2020

 

Week 9 DNA isolation, PCR, and gel electrophoresis (16 points)

 

Purpose:

These activities will familiarize you with the procedure for isolation of DNA from cheek cells, the amplification (copying) of specific regions of DNA through polymerase chain reaction (PCR), and the separation and visualization of DNA using gel electrophoresis.  These simulations are fairly realistic and representative of the procedures that we would have completed in our two-week lab, but can be completed in less time in these simulations.

Task:

Follow the instructions provided below and work through the following three exercises. Contact your instructor as soon as possible if you cannot get the websites to work; alternate arrangements will require some scheduling time!  Please type your answers to the post-lab questions in bold in this document, save it as a Word document, and upload it to Canvas by the due date provided by your instructor.

Criteria for Success:

The successful student will allow themselves plenty of time to read the information provided in each simulation and complete these three simulations.  Your grade will reflect your ability to follow the instructions within the exercises and interpret the information provided.

 

Part1:  DNA Isolation Simulation Instructions.

Complete the University of Utah’s DNA extraction simulation.

• This will require Flash.
• Give yourself plenty of time to follow the DNA extraction simulation provided, read the information throughout the simulation, and to analyze the data.
• To start the simulation, scroll down a bit and click the arrow that says “start lab.”
• Click on “next” arrows at the bottom right of the panels in order to work your way through the simulation.
• You will need to do some clicking and dragging to move various pieces of equipment during this simulation.

Post-lab questions for Part 1 (DNA Isolation):

Answer the following questions:

 

1. Using the simulation as a guide but using your own words to explain the procedure, answer the following: (2 points)
   1. How were the cheek cells collected from the “person” in the simulation?
   2. What had to happen to the cheek cells in order to release the DNA from the nucleus of the cells?
   3. After adding salts to the solution, it needed to be centrifuged. What happened during the centrifugation step?
   4. Isopropyl alcohol was then added to the solution containing the DNA. Why was the isopropyl alcohol added?
2. Identify four potential uses for the isolated DNA: (1 point)
   1. Two uses of DNA for the identification of a specific person:
   2. Two uses of DNA that do not involved the identification of a specific person (Note that you may need to think about topics that were discussed earlier in the class):

 

 

Part2:  Polymerase Chain Reaction (PCR) Simulation Instructions.

Complete the University of Utah’s PCR Virtual Lab simulation.

• As above, this will require Flash.
• Give yourself plenty of time to follow the PCR simulation (remembering that this is much faster than if we were to do PCR in the lab), read the information throughout the simulation, and to analyze the data.
• Before starting the PCR simulations, scroll down and read “what are these things doing in my PCR reaction?”
• To start the PCR simulation, scroll down a bit and click the blue “begin.”
• Click on “next” arrows at the bottom right of the panels in order to work your way through the simulation.
• You will need to do some clicking and dragging to move various pieces of equipment during this simulation.

 

Post-lab questions for Part 2 (PCR):

Answer the following questions:

 

3. Read “what are these things doing in my PCR reaction?” on the University of Utah’s PCR simulation webpage before starting the actual simulation activity. Answer the following: (2 point)
   1. What is the function of the primers in a PCR reaction?
   2. What is the function of DNA polymerase in a PCR reaction?
   3. What specific DNA polymerase is used in PCR and why is it necessary to use this specific version of the enzyme?
   4. What are nucleotides and what is the function of nucleotides in a PCR reaction?

 

4. Use the PCR simulation as a guide, read the provided instructions, click “okay,” “next,” “start,” or the arrow and do some clicking and dragging to move various pieces of equipment during this simulation. Using your own words to explain the procedure, answer the following: (2 point)
   1. What is the overall goal of PCR?
   2. When you are assembling the PCR reaction, what are the five solutions that the simulation instructs you to add to the PCR tubes one-by-one?
   3. In degrees Celsius, how hot does the thermocycler heat to? What is the equivalent in degrees Fahrenheit?
   4. What happens to the DNA every time the DNA is heated to the temperature noted in 4c?
   5. How many cycles of the thermocycler were completed in this simulation?

 

Part3:  Gel Electrophoresis Simulation Instructions.

Complete the University of Utah’s gel electrophoresis simulation.

• As above, this will require Flash.
• Give yourself plenty of time to follow the gel electrophoresis simulation (remembering that this is much faster than if we were to run a gel in the lab), read the information throughout the simulation, and to analyze the data.
• To start the gel electrophoresis simulation, scroll down a bit and click the “forward” arrow.
• Click on “forward” arrows at the bottom right of the panels in order to work your way through the simulation.
• You will need to do some clicking and dragging to move various pieces of equipment during this simulation.

 

Post-lab questions for Part 3 (gel electrophoresis):

Please answer the following questions:

 

5. Use the gel electrophoresis simulation as a guide, read the provided instructions, click the “forward” arrow to move through the simulation, and do some clicking and dragging to move various pieces of equipment during this simulation. Using your own words to explain the procedure, answer the following: (2 points)
   1. How do we sort and measure the DNA strands in your tube, even though you can’t see or touch the DNA?
   2. How does the simulation describe the gel that sorts the DNA strands?
   3. Where do we put the DNA samples?
   4. What charge does DNA have?

 

6. Use the simulation to complete the “making a gel” portion of the simulation that takes place in the Gel Electrophoresis Laboratory. Continue to do some clicking and dragging to move various pieces of equipment during this simulation and click the “forward” arrow to move through the simulation.  Using your own words to explain the procedure, answer the following: (2 points)
   1. What solid material is used to make a gel?
   2. In this simulation, what is that powder isolated from?
   3. What piece of equipment do we use to heat the gel to get it to go into solution?
   4. When we run the gel, is it dry on the countertop or it is “wet” when submerged under a buffer?

 

7. Continue using the gel electrophoresis simulation as a guide. Using your own words to explain the procedure, answer the following: (2 points)
   1. What piece of equipment is used to transfer the DNA from one tube to another?
   2. Your PCR sample is loaded into one lane. What is loaded into a separate lane on the same gel?
   3. What is applied to the gel in order to move the DNA through the gel?
   4. When the gel is “running,” what can be seen coming off of the electrodes at both ends of the gel box?

 

8. Continue using the gel electrophoresis simulation as a guide. Using your own words to explain the procedure, answer the following: (1 points)
   1. What is the blue substance that we can see moving through the gel with our naked eye?
   2. In this simulation, the chemical ethidium bromide is used to bind to DNA. Because this is a known carcinogen (we can now use safer chemicals in our lab at CBC), students would need to make sure to wear what piece of protective equipment?
   3. When ethidium bromide is exposed to a specific kind of light, it fluoresces. What kind of light box used to make the DNA stained with ethidium bromide glow?
   4. In this simulation, the light in this gel box is damaging to your eyes (we can now use safer chemicals in our lab at CBC), students would need to make sure to wear what piece of protective equipment?

 

9. Within the simulation, view the DNA on the gel that results from gel electrophoresis. Using your own words to explain the procedure, answer the following: (1 point)
   1. In this simulation, are the wells at the top of the gel or at the bottom of the gel?
   2. Do shorter or longer fragments move more quickly through the gel?
   3. What are the approximate sizes of the three DNA fragments seen in this simulation?
   4. What is the unit that we use to indicate the length of each DNA fragment?

 

10. Analyze the gel used in the Crime Scene Investigator PCR Basics Kit from BioRad below.

 

Answer the following: (1 point)

1. Compare the banding patterns In the gel from the “Crime Scene Investigator PCR Basics Kit” above.  Which suspect below likely committed the crime at the crime scene?
2. Other people may be at the scene of the crime for legitimate reasons. Who else’s DNA would you likely want to check to make sure that they did not contaminate the crime scene?
3. Do criminals need to leave a large DNA sample behind, or can we isolate DNA from single cells and identify criminals from a very small DNA samples?
4. If your criminal was a monozygotic twin, can you use PCR alone to tell which twin committed the crime? Why or why not?

 

 

As a purely optional exercise (for instance, if you want a visual activity that you could do with your kids), if you would also like to isolate DNA at home check out the University of Utah’s “How to Extract DNA from Anything.”

 

 

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