PCR/Primer Design and Cloning Now that you have learned a little bit about the genetics of the unknown organism you have been asked to create a real-time PCR assay to detect the unknown in other patient's blood samples using the gene that was amplified by the clinical lab. To create your standards for your real-time PCR assay you need to clone your DNA fragment into a plasmid (pUC19) using standard PCR techniques. 2) Using the 825 bp sequence for your unknown fragment, please create a primer pair that will amplify a 500 base pair fragment of the gene. Additionally, please add restriction sites to the ends of your primers. Please provide the following: • Sequences of the primers • Primer Tm • The location of the primers in the sequence • The restriction sites you used. Remember: Check to make sure that the restriction sites will only cut the primers and are not found anywhere else in the DNA sequence but are found in pUC19.

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PCR/Primer Design and Cloning Now that you have learned a little bit about the genetics of the unknown organism you have been asked to create a real-time PCR assay to detect the unknown in other patient's blood samples using the gene that was amplified by the clinical lab. To create your standards for your real-time PC