Site-directed mutagenesis: Difference between revisions
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#Run a PCR reaction with a proof-reading, non-displacing polymerase such as ''PFU'' DNA polymerase. This results in nicked circular strands of the plasmid. | #Run a PCR reaction with a proof-reading, non-displacing polymerase such as ''PFU'' DNA polymerase. This results in nicked circular strands of the plasmid. | ||
#Cut up the template DNA with ''Dpn''1. | #Cut up the template DNA with ''Dpn''1. | ||
#Transform the circular nicked DNA into a strain such as XL1-Blue. These cells will repair the nicks. | #Transform the circular nicked DNA into a highly competent strain such as XL1-Blue. These cells will repair the nicks. | ||
#Select colonies with the correct DNA. | #Select colonies with the correct DNA. | ||
==Specific Protocols== | ==Specific Protocols== | ||
*[[Endy:Site-directed Mutagenesis]] | *[[Endy:Site-directed Mutagenesis]] | ||
Revision as of 11:52, 13 July 2005
General Information
Site-directed mutagenesis can be used to change particular base pairs in a piece of DNA. There are a number of methods for achieving this. The approach described here is adapted from the Stratagene site-directed mutagenesis kit, the manual can be found here. Even when using a kit it will be necessary to design primers that are suitable for the specific changes you want to make to your DNA. Most of the contents of the kit can be found in your favorite labs stocks so you may not need to buy the kit itself.
General Procedure
- Design forward and reverse primers that will bind to the region of DNA you want to mutate but that contain the modifications you wish to make.
- Run a PCR reaction with a proof-reading, non-displacing polymerase such as PFU DNA polymerase. This results in nicked circular strands of the plasmid.
- Cut up the template DNA with Dpn1.
- Transform the circular nicked DNA into a highly competent strain such as XL1-Blue. These cells will repair the nicks.
- Select colonies with the correct DNA.
Specific Protocols
Notes
- This protocol is at a very early stage of development. Any contributions welcome!
