Nucleic Acids and Oligonucleotides

Mutagenic primer Selection primer

Both mutagenic and selection primers must anneal to the same strand of the target DNA, and the 5' end of each primer must be phosphorylated. The oligonucleotide primers should be purified by FPLC or PAGE before use (please see Chapter 10, Protocol 1 or Chapter 10, Protocol 5).

Several companies (CLONTECH, Pharmacia) sell selection primers and some companies (e.g., Pharmacia) also market pairs of"toggle" primers that are used for forward and reverse conversion of restriction sites and allow sequential rounds of mutagenesis without subcloning of the template.

Plasmid DNA

Closed circular plasmid DNA, purified either by chromatography through a commercial resin or by the alkaline lysis method (please see Chapter 1, Protocol 2 or Chapter 1, Protocol 9).

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