Homemade site directed mutagenesis of whole plasmids.
Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.
Laible
Mark
M
Department of Biology, Johannes Gutenberg-University Mainz, Germany.
Boonrod
Kajohn
K
D004926:Escherichia coli / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D014169:Transformation, Bacterial