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.