Difference between revisions of "Lidstrom:Site-Directed Mutagenesis"
Latest revision as of 05:56, 12 June 2014
Back to Protocols
- 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
- 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.