Lidstrom:QuikChange Site-Directed Mutagenesis: Difference between revisions
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* The marketing term "lightning" refers to an improved Dpn1, that degrades template (unmutated template) more efficiently. | * 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. | * 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 == | == Two (main) QuikChange kits are available == | ||
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** Can be used for mutations contained in 1 primer, but this kit is more expensive than the single kit and likely has lower efficiency. | ** 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: | * Both kits: | ||
** come with chemically competent XL10-Gold. This is perhaps the most valuable part of the kit. | ** come with chemically competent XL10-Gold. This is perhaps the most valuable part of the kit. | ||
== 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|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.) | ||
Revision as of 14:32, 16 September 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.
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.)
