Homemade Site Directed Mutagenesis of Whole Plasmids

Summary

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Abstract

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.

Protocol

Principle of Method:

The site directed mutagenesis of whole plasmids explained in this video is a mutagenesis method which allows you to alter a cloned target gene by substitution, deletion or insertion of a few bases directly into a plasmid. It works by amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation in form of mismatches to the original plasmid, are integrated into the newly synthesized plasmid. After removal of the original plasmid from the reaction the mutated plasmid gets transformed into E. coli. The following steps are for screening p....

Discussion

Site directed mutagenesis is a mutagenesis-method which provides a fast way to mutate a gene carried by a plasmid. The whole reaction can be done in only one day. With this method, it will need only a pair of complimentary primers carrying the desired mutations and a proofreading polymerase such as Pfu-Polymerase. The newly synthesized plasmid can be separated from the parental plasmid by digesting the reaction with the restriction enzyme DpnI. This enzyme digests only the methylated DNA. Therefore only the newly synthes.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pfu polymerase (recombinant or native)	Fermentas	EP0571 or EP0501
dNTP mix 10 mM	Fermentas	R0191
DpnI or DpnI Fast digest	Fermentas	ER1701
primers	Invitrogen		desalted