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. 1983 Aug 11;11(15):5103–5112. doi: 10.1093/nar/11.15.5103

Efficient site-directed mutagenesis by simultaneous use of two primers.

K Norris, F Norris, L Christiansen, N Fiil
PMCID: PMC326240  PMID: 6308572

Abstract

A rapid and efficient procedure for site specific mutagenesis is described. A double primed synthesis with a 17-mer mismatch primer and a "universal" 15-mer M13 sequencing primer was used to introduce a T to A transversion into an ompF signal peptide gene cloned in the M13mp8 vector. The two primers were annealed to the circular single stranded M13 template. After a short extension and ligation reaction, a double stranded restriction fragment containing the mismatch (ompF*/EcoR1-SalI) was cut out of the partly single stranded circular DNA and inserted into pBR322. 42% of the E.coli transformants harboured plasmid with the desired mutation, which could be detected by the appearance of a new restriction site (MboII) and by dot blot hybridization of plasmid DNA with the 32P-labeled 17-mer.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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