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Question: If your colleague sets the denaturing, annealing and extension temperatures in his PCR experiment…

Question: If your colleague sets the denaturing, annealing and extension temperatures in his PCR experiment...

If your colleague sets the denaturing, annealing and extension temperatures in his PCR experiment to 96, 40, and 72 degree C, respectively, do you think he will obtain amplification of the 60 base pair sequence that he wants? Explain why or why not. If your colleague changes the denaturing, annealing and extension temperatures in his PCR experiment to 96, 15, and 72 degree C, respectively, would this experiment yield an improvement over the results he obtained in part i)? Explain why or why not. Generally speaking, most PCR reactions will have a final concentration of template DNA and primers at Tilde1-100 pM (template) and Tilde1 mu M (primers). Your colleague’s PCR experiment is scheduled to run for 2 hours, but he is impatient and he wants to speed up the amplification. His plan is to increase his primer concentration by 10 fold so that his sequence will amplify faster. Do you think his plan will work? Explain why or why not. Your friend finally gives up and decides to synthesize the sequence using Gibson assembly as opposed to amplifying with PCR. Starting with the primer below, provide the sequence of additional 10-base pair long primers required to synthesize the total 60 bp region. For this question, all primers have to be 10 bases long and you can assume you need 5 base pairs of overlap between primers.

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