Molecular Biology Experimental Design. Restriction enzyme, primer sequence

For the final paper, include everything that was in the approved introduction, plus design a cloning strategy detailing the primers used to PCR clone, restriction enzymes, screening techniques and protein purification method to be used. The text should resemble a materials and methods section of a research paper with enough detail that a researcher in another lab could duplicate the experiment. References or commercial sources for vectors, purifications kits, etc. are required. The text should be concise and no more than five single-spaced, typed written pages (plus additional pages for figures and references).

PCR primers should be shown and the sites to which the primers anneal should be highlighted and denoted on the GenBank file, as should the start and stop codons of your gene of interest. Also indicate how you know that your gene is in the correct reading frame and orientation. Include a brief description of which cellular strain you will use and why, how you will screen for positive clones and confirm the correct sequence of the insert, how you will induce protein expression, and how you will purify the protein. You should use the checklist and the paper should have the same section headings as found in the checklist.

In addition to the highlighted GenBank file and the vector map, include a written flow chart of the cloning process that you will use.