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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?

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There are a number of primer testing tools that will compare primer sequences against the genome to ensure specificity, or help you design specific primers to amplify a specific region. One place to start is NCBI Primer BLAST; the linked page explains how to use it to generate primers to amplify a specific gene.

Doing PCR on genomic DNA can be a bit tricky, though, since you have to deal with a lot of confounding factors like DNA accessibility, contamination, and PCR inhibitors that get through your purification procedure. In addition, genes often contain huge introns, so you're going to end up doing PCR on an extremely long template (which is very hard or even impossible, depending on the size of the gene) and you'll end up with the whole gene sequence with introns which is not what you need in order to express it as a transgene.

Instead, I'd recommend purchasing a cDNA clone of your gene of interest and amplifying it from that. cDNA is made from the spliced mRNA sequence, so it will be a much more managable length and will prevent problems with alternate splicing.

If you go to the NCBI page for a gene, on the right sidebar under "Related Information" you'll see a link to Order cDNA clone. This will give you a few options to purchase a purified plasmid containing that gene for around \$60 - \$90. It will be much easier to work with this plasmid than genomic DNA and it will definitely save you time and money, especially if you're still inexperienced at molecular cloning.


Of course, it's not 2003 anymore and there's a good argument to be made that the whole idea of doing molecular cloning yourself is a waste of time and money. If your budget allows it, the easiest and fastest way is to pay a company to synthesize your gene for you. Genewiz, for example, offers snythesis for $0.28/bp, but you can probably get a better deal if you shop around.

The advantage of synthesis is that you can get the exact sequence you want cloned into exactly the right vector done perfectly the first time. If you don't have experience with it, trying to clone a gene yourself will take longer and cost more money than you think. It's better to just get it synthesized so you can move onto your real experiment.

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  • $\begingroup$ I was going to suggest getting a synthetic gene, but now I’ll just second your answer. Some (most?) companies will even correct for codon bias, etc if you so desire. $\endgroup$ – canadianer Dec 21 '18 at 20:25
  • $\begingroup$ @canadianer That's a good point. Since the asker mentions "engineering applications", they're probably going to care about getting good levels of expression. $\endgroup$ – divibisan Dec 21 '18 at 20:42
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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.

No. The point of the primers is that they should be long enough to bind to one specific locus (or at least, there is only one place where the two primers bind close enough to each other and in the right orientation for PCR to happen)

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