An improved PCR-based method for site directed mutagenesis using megaprimers

An improved protocol for site-directed mutagenesis based on the two-step polymerase chain reaction (PCR) megaprimer method is described. Compared to previously published protocols, the protocol described in this article ensures consistently a success rate of at least 85% with essentially no introduc...

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Veröffentlicht in:Molecular and cellular probes 1998-12, Vol.12 (6), p.345-348
Hauptverfasser: Brøns-Poulsen, J, Petersen, N.E, Hørder, M, Kristiansen, K
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Sprache:eng
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Zusammenfassung:An improved protocol for site-directed mutagenesis based on the two-step polymerase chain reaction (PCR) megaprimer method is described. Compared to previously published protocols, the protocol described in this article ensures consistently a success rate of at least 85% with essentially no introduction of unwanted secondary mutations. The essential features of this protocol include an optimization of the template-primer amounts and ratio that allows the use of a reduced number of PCR cycles and the use of proof-reading thermostable DNA polymerases.
ISSN:0890-8508
1096-1194
DOI:10.1006/mcpr.1998.0187