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Wk 3 - Virtual Bacterial Identification Lab Experience: PCR Amplification Lab Handout Part I: Respond to the following questions: 1. What does “PCR” stand for and what is the purpose of PCR? Polymerase Chain Reaction, Create multiple copies of a segment of DNA 2. What does the Master Mix contain? Water 3. What are primers? Why is a primer added? Primers are small pieces of DNA that bind to specific sequences. It is added to replicate the DNA 4. Once the primers bind, what occurs next? Once the primer binds DNA polymerase extend the DNA from the 5’ end to this 3’ end. The primers are selected so that as the two new copies are made they overlap in the region of interest. 5. What does "highly conserved" mean? It means that there are some parts of the Jean that are extremely similar among different species. 6. Why are highly conserved regions important in this lab? Because the universal primers bind to the highly conversed regions of the genes. This is so that they can be used to copy DNA from a variety of species of bacteria. 7. What does "highly variable" mean? It means that they are DNA that are extremely different among different species 8. Why are highly variable regions important in this lab? The variable regions that differ are used from identification. 9. What is missing in the negative control tube? Solution of 16S rDNA in the green capped bottle 10. What is present in the positive control tube that is not in the negative control tube? What is present in the positive control tube is the Solution of 16S rDNA in the green capped bottle. And the negative control tube reaction contains sterile deionized water. Both reactions have the PCR master mix solution.

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Syllabus SCI/250 v4 Page 2 of 2

Part II: Run the PCR and watch the virtual lab animation. Respond to the following questions: 1. List each step of a PCR cycle, the temperature, and the duration (time). ○ Melt: 95°C 30 seconds ○ Anneal: 60°C 30 seconds ○ Extend: 72°C 45 seconds 11. Describe what happens during each of the steps in one or two sentences. - Initialization/ Incubation: In this first step the sample is heated to 95°C for 10 minutes. This is the start of the PCR reaction. In this step it is helped to activate the polymerases thus allowing for melting to occur. -

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Melting/ Denaturation: Each cycle, the first step of melting occurs. The “PCR reaction mixture to 95°C for 30 seconds” this causes it to melt/denature both your DNA and primers. Doing that allows them to anneal to each other in the next step. Annealing: In this step the vial is cooled to 60°C because “the primers cannot bind to the DNA strands at such a high temperature” (Virtual Lab). At 60°C, “the primers bind (anneal) to the single-stranded DNA. (The reason the two separated strands of DNA do not reanneal is that there is a large excess of primers in the solution; therefore, it's more likely for the DNA strands to bind to the primers instead of to each other.)” (Virtual Lab).

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Extend/ Elongation: “The final step (Extend) is to allow the DNA polymerase to extend the copy DNA strand by raising the temperature to 70°C for 45 seconds.” (Virtual Lab). At the end of this step the new double-stranded pieces of DNA will be created, consisting of both template and new DNA.

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Repeat if wanted or needed

12. After eight cycles, how many copies of the desired DNA have been synthesized? 240 13. After 30 cycles? 1 million

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