Lidstrom:Site-Directed Mutagenesis: Difference between revisions
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(New page: Back to Protocols ==Key Concepts== *There are several ways to change bases. Qucick-Change is the fast and standard way. *Some protocols use ligase. *Some protocol...) |
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==Key Concepts== | ==Key Concepts== | ||
*There are several ways to change bases. | *There are several ways to change bases. QuickChange is the fast and standard way. | ||
** Our lab has some protocol notes [[Lidstrom:QuikChange_Site-Directed_Mutagenesis|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 [http://www.genomics.agilent.com/primerDesignProgram.jsp 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 use ligase. | ||
*Some protocols allow you to do more than one change simultaneously. | *Some protocols allow you to do more than one change simultaneously. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2629768/pdf/1472-6750-8-91.pdf Here] is an excellent article Amanda found about doing multiple mutations with QuickChange | ||
==Kunkel Mutagenesis== | ==Kunkel Mutagenesis== | ||
*Justin Siegel at the Baker lab is our local expert. The [http://2010.igem.org/Team:Washington/Protocols/Kunkel UW iGem] team uses this method. | *Justin Siegel at the Baker lab is our local expert. The [http://2010.igem.org/Team:Washington/Protocols/Kunkel 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. | *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. | ||
Latest revision as of 06:56, 12 June 2014
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.
