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Keywords:

  • Site-directed mutagenesis;
  • troubleshooting;
  • controls

Abstract

By shifting pedagogical goals from obtaining successful mutations to teaching students critical troubleshooting skills, it has been possible to introduce site-directed mutagenesis into an undergraduate teaching laboratory. Described in this study is an inexpensive laboratory exercise in which students follow a slightly modified version of Stratagene's QuikChange site-directed mutagenesis kit to effect a single amino acid change in ribonuclease Sa. From the students' perspective, they are performing an authentic mutagenesis reaction. However, by judicious substitution of most of the reagents, the exercise has been made economically feasible for implementation in large classes while still providing students the opportunity to learn not only the underlying theory of site-directed mutagenesis but also a host of associated concepts such as transcription, translation, PCR, DNA methylation, restriction digests, transformations, and blue/white screening. Just as importantly, this exercise simulates a wide range of mutagenesis failures, allowing students the opportunity to troubleshoot an experiment by carefully analyzing the results of positive and negative controls, thus helping to develop analytical thinking skills in a way that simply would not happen if students' experiments “worked.”