For background I am interested in studying engineering applications of a specific protein, which is not commercially available. My end goal is to express the gene for the protein in bacterial cells, most likely E. Coli, if that is a feasible procedure. I am mostly familiar the process of forming recombinant DNA, and though our lab does not specialize in Biology, we will be working with another lab in our Biomedical department which can provide necessary equipment and some advise.
The last time I did a lab where we expressed a new gene in E. Coli cells was a biotechnology class in high school, though much of the set up was done for us. I recall that our first step was amplifying the desired DNA fragment through PCR, but what I don't know is how the fragment was initially isolated. I know the primers help by essentially setting the start and end positions for the DNA cloning, but if you were to use an entire chromosome that contains the desired gene, it would seem likely that there would be many possible sites that the primers could attach besides the desired region. So if the DNA sequence for the protein I want is already known, how would I get a fragment that contains the gene I want?
Additionally, how difficult will this process be? Since the end goal is actually to study the protein, I do not want this step to turn into a full thesis on its own. For some specific proteins, the process seems well documented and can be done using relatively common materials and by following simple instructions, but how much should I expect the procedure to diverge for attempting this with gene that has not been specifically done before?