Every PCR requires two short DNA molecules, called primers, that are specific to the gene you are trying to amplify. The sequence of these primers is determined by the sequence of the DNA that you are trying to amplify. In the previous section we were able to find our gene in the NCBI database and we found the sequence of that gene.
DNA sequences in the NCBI database are always published from the 5’ to 3’ direction, but remember DNA is composed of a coding strand and a complementary template strand running 3’ to 5’. Two primers are designed for the PCR; a forward primer that extends one strand from the start codon to the stop codon and a reverse primer that extends from the stop codon to the start codon. That looks something like this:
A tutorial on how to design primers is found here: PCR Primer Design
There is also a video tutorial on the next page.
There are some general concepts to use when designing primers:
Remember: The next step is to come into the lab to practice with the materials, view a demonstration, or to even just ask questions. Also, if you are ready to take the lab final, please inform your instructor. The lab final will be performed by yourself and will be graded with a rubric.