Lidstrom:QuikChange Site-Directed Mutagenesis: Difference between revisions

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* 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|JM's]] use of one primer to introduce 24 different mutations.  (The first colony screened had the desired mutation in 90+% of the sequenced plasmids and the efficiency was quite high.)
* 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|JM's]] use of one primer to introduce 24 different mutations.  (The first colony screened had the desired mutation in 90+% of the sequenced plasmids and the efficiency was quite high.)
* From the manual:
** Thaw the dNTP mix once, prepare single-use aliquots, and store the aliquots at –20°C. Do not subject the dNTP mix to multiple freeze-thaw cycles
* Make your kit last longer by doing 8uL reactions, not 25uL.  You only transform 1uL per reaction so this is always plenty.

Revision as of 14:37, 16 September 2014

Back to Protocols

Manuals


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.

Quik Solution

  • Quik Solution is probably pure DMSO.

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 JM's use of one primer to introduce 24 different mutations. (The first colony screened had the desired mutation in 90+% of the sequenced plasmids and the efficiency was quite high.)
  • From the manual:
    • Thaw the dNTP mix once, prepare single-use aliquots, and store the aliquots at –20°C. Do not subject the dNTP mix to multiple freeze-thaw cycles
  • Make your kit last longer by doing 8uL reactions, not 25uL. You only transform 1uL per reaction so this is always plenty.
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