Lidstrom:Site-Directed Mutagenesis

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Back to Protocols

Key Concepts

  • There are several ways to change bases. QuickChange is the fast and standard way.
    • Our lab has some protocol notes here
    • QuikChange is an Agilent product (acquired Stratagene), and uses mutagenic primers with polymerase and ligase to introduce new mutations, then Dpn1 to degrade template plasmids.
    • There is QuikChange Lightning for mutations contained in one priming site, and QuikChange Mult Lightning for doing mutations contained in multiple priming sites simultaneously.
    • Agilent provides a free web tool for primer design. You have to make an account to use it, but it is nice to use.
  • Some protocols use ligase.
  • Some protocols allow you to do more than one change simultaneously. Here is an excellent article Amanda found about doing multiple mutations with QuickChange

Kunkel Mutagenesis

  • Justin Siegel at the Baker lab is our local expert. The UW iGem team uses this method.
  • Our lab (at least Amanda and Janet) have not tried it yet. Our understanding is that it is for more substantial changes than just single amino acid changes.
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