Lidstrom:Site-Directed Mutagenesis

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Key Concepts)
Current revision (08:56, 12 June 2014) (view source)
 
Line 2: Line 2:
==Key Concepts==
==Key Concepts==
-
*There are several ways to change bases.  QuickChange is the fast and standard way.  
+
*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.  
** 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.

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.
Personal tools