Lidstrom:QuikChange Site-Directed Mutagenesis

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== Tips ==
== Tips ==
* Though the lightning Dpn1 enzyme claims to be effective in 5 minutes, you might as well incubate it for 30 minutes, just in case it lowers your unmutated background further.
* Though the lightning Dpn1 enzyme claims to be effective in 5 minutes, you might as well incubate it for 30 minutes, just in case it lowers your unmutated background further.
 +
* If you want to use one priming site to introduce mutations, it is about the same price to order 1 oligo and do it with the multi kit, or order 1 pair of complementary oligos and do it with the non-multi kit.  If ordering less primers is appealing, go for it.  A small decrease in efficiency might be seen, but it shouldn't be a problem based on *'''[[User:Janet B. Matsen|Janet Matsen]] 10:29, 12 June 2014 (EDT)''':

Revision as of 10:29, 12 June 2014

Back to Protocols

QuikChange Basics

  • You can buy a kit from Agilent or use your own ingredients.
  • The marketing term "lightning" refers to an improved Dpn1, that degrades template (unmutated template) more efficiently.
  • If you buy the kits, 10uL rxns are plenty (this is ~4x less than the protocol suggests) and transformations using 10uL of competent cells are suitable. If you are introducing mutations with multiple priming sites, you could consider transforming more.

Two (main) QuikChange kits are available

  • QuikChange Lightning.
    • For mutations contained within a single primer
  • QuikChange Lightning Multi
    • For mutations contained in >1 primer
    • Can be used for mutations contained in 1 primer, but this kit is more expensive than the single kit and likely has lower efficiency.
  • Both kits:
    • come with chemically competent XL10-Gold. This is perhaps the most valuable part of the kit.

Tips

  • Though the lightning Dpn1 enzyme claims to be effective in 5 minutes, you might as well incubate it for 30 minutes, just in case it lowers your unmutated background further.
  • If you want to use one priming site to introduce mutations, it is about the same price to order 1 oligo and do it with the multi kit, or order 1 pair of complementary oligos and do it with the non-multi kit. If ordering less primers is appealing, go for it. A small decrease in efficiency might be seen, but it shouldn't be a problem based on *Janet Matsen 10:29, 12 June 2014 (EDT):
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