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The 'megaprimer' method of site-directed mutagenesis uses three oligonucleotide primers and two rounds of polymerase chain reaction (PCR)1. One oligonucleotides is mutagenic; the others are forward and reverse primers that lie upstream and downstream from the binding site for the mutagenic oligonucleotide. The mutagenic primer and the nearer of the external primers are used in the first PCR to generate and amplify a mutated fragment of DNA. This amplified fragment—the megaprimer—is used in the second PCR in conjunction with the remaining external primer to amplify a longer region of the template DNA. This protocol is based on a method that uses forward and reverse external primers with significantly different melting temperatures (Tm)2.
Southern transfer and hybridization1 is used to study how genes are organized within genomes by mapping restriction sites in and around segments of genomic DNA. This protocol describes the first stages of Southern blotting: digestion of genomic DNA with restriction enzymes, separation of the resulting fragments by gel electrophoresis, and capillary transfer of the denatured fragments to a membrane2.