Site-directed mutagenesis

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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.

General Procedure

  1. 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.
  2. 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.
  3. Cut up the template DNA with Dpn1.
  4. Transform the circular nicked DNA into a strain such as XL1-Blue. These cells will repair the nicks.
  5. Select colonies with the correct DNA.

Specific Protocols

Notes

  • This protocol is at a very early stage of development. Any contributions welcome!

References

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