PCR is a powerful tool used to confirm presence of DNA sequences and PCR primer design is key to success in DNA amplification. Students often struggle with aspects of primer design such as figuring out where primers should bind, on what DNA strand the primers should bind to, and how to ensure replication of their region of interest. To help students with these concepts, I have incorporated primer design, group evaluation, and then use of student designed primers into a third-year molecular techniques laboratory. Students use Agrobacterium rhizogenes containing vector pCAMGUSGFP to transform soybean roots. To confirm success, they use fluorescence microscopy and GUS staining to provide phenotypic evidence of transformation. I then have students design primers to amplify either GUS or GFP as neither of these sequences are found in plant cells. These primer sequences are submitted to the entire class anonymously and then students work in groups to evaluate each pair of primers dealing with questions such as ?Do the primers bind? Do they amplify what they are supposed to? Do they fulfill conditions for successful primers ? eg length, GC content?. Students are also asked to predict the results from PCR of transformed plants by drawing the results of agarose gel electrophoresis. Students then submit their top three primer pairs along with their group evaluations. We order the top ranked primers and use them in PCRs of root tissue showing evidence via microscopy and staining of transformation to confirm transformation success. Students then reflect on their choice of primers and their understanding of primer design in a written assessment.
Keywords: PCR, soybeans, transformation, primers
University of Manitoba (2025)
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