A rapid and versatile site-directed method of mutagenesis for double-stranded plasmid DNA
Related Experiment Video
Updated: Sep 4, 2026

Homemade Site Directed Mutagenesis of Whole Plasmids
Published on: May 11, 2009
A V Bellini1, F de Ferra, G Grandi
1ENIRICERCHE S.p.A., Department of Molecular Biology, Milan, Italy.
Abstract:
This paper describes a new method for site-directed mutagenesis which allows mutations by deletion, insertion or substitution of large fragments of DNA with more than 50% efficiency and does not require subcloning in a single-stranded (ss) DNA vehicle. The site of mutagenesis is removed from a linearized plasmid DNA by BAL 31 digestion, ss DNA regions are generated by limited exonuclease treatment and the mutated target site is reconstituted by annealing of the plasmid DNA to a 35-70 nucleotide long mutated ss oligodeoxynucleotide containing the desired mutation. The circularized plasmid is finally used to transform directly Escherichia coli competent cells.
More Related Videos
Related Concept Videos
Conservative Site-specific Recombination and Phase Variation
The recognition sites for Cre recombinase called LoxP...
DNA Isolation
DNA Bacteriophages

