Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is useful to study molecular interactions, such as RNA-RNA, and RNA protein interactions. This protocol describes how to perform site-directed mutagenesis with a two or 3-step PCR approach. The main advantages of the method are the diversity of different mutations that can be incorporated, as well as a relative ease and cost for doing so.

To begin, use the wild-type DNA template and primer pairs two, one, and two, three, specific for 2-step, or primer pairs three, one, and three, four, specific for 3-step PCR to obtain PCR product one. To validate PCR products by agarose gel electrophoresis, make a 2%agarose gel by dissolving two grams of agarose in 100 milliliters of 1X TAE buffer, using a microwave oven. Add ethidium bromide at a final concentration of 0.5 microgram per milliliter.