Simple version of "megaprimer" PCR for site-directed mutagenesis.

use in restriction-enzyme analysis. This procedure is cost-effective because it saves the use of expensive miniprep kits for use with only positively identified recombinant clones and limits typically time-consuming miniprep steps. Our method can be accomplished in a few minutes in two microcentrifuge tubes with minimal enzyme, expense, no phenol/chloroform extractions and no ethanol precipitations. We routinely prepare overnight cultures on day one, perform the microprep isolation and restriction enzyme analysis the following morning and then perform a miniprep isolation (e.g., using S.N.A.P. Nucleic Acid Isolation Kit; Invitrogen, Carlsbad, CA, USA) of positive recombinants in the afternooon yielding DNA that is ready for sequencing, transfection, in vitro transcription/translation, PCR, restriction mapping, ligation, or transformation.