Lidstrom:Site-Directed Mutagenesis

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(Key Concepts)
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==Key Concepts==
==Key Concepts==
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*There are several ways to change bases.  QuickChange is the fast and standard way.  
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*There are several ways to change bases.  QuickChange is the fast and standard way.
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** 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.  
** 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.
** There is QuikChange Lightning for mutations contained in one priming site, and QuikChange Mult Lightning for doing mutations contained in multiple priming sites simultaneously.
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** 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.  [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
*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

Current revision

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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