1. You are given a pair of primers that has been shown amplify a 125 bp DNA of interest specifically by a end point PCR- as there was only one PCR product with a size of 125 bp detected by agarose gel electrophoresis. How would you further develop this end-point PCR to be a quantitative real-time PCR? Please provide a rationale for each step that you choose to further develop the assay and what is the alternative if your first choice is not working.
2.You have been asked to develop a monoclonal antibody against the human His-tagged Casc4 recombinant protein that you purified from the expression construct that you designed earlier in the semester (assume the protein is soluble).
a.) Outline a brief protocol for generating this monoclonal antibody using mice as the host species.
b.) What is a method for purifying this antibody from culture supernatant?
3. Zach wants to analyze the expression of transferrin receptor (a surface transmembrane protein) in an epithelial cell line with and without treatment using a certain drug. a.) Outline a brief protocol for obtaining these results. b.) Why did you choose this method?