Lidstrom:QuikChange Site-Directed Mutagenesis: Difference between revisions
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Back to [[Lidstrom:Protocols|Protocols]] | Back to [[Lidstrom:Protocols|Protocols]] | ||
== Manuals == | |||
* [http://www.chem.agilent.com/library/usermanuals/Public/210518.pdf QuikChange II]: use 2 oligos (reverse compliments) and only do mutations at one priming site | |||
* [http://www.chem.agilent.com/library/usermanuals/Public/210514.pdf QuikChange II Lightning]: use 1 oligo, and can introduce mutations at multiple sites | |||
==QuikChange Basics == | ==QuikChange Basics == | ||
| Line 5: | Line 9: | ||
* 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 == | ||
| Line 13: | Line 20: | ||
** 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. | ||
== Primer Design == | |||
* Use online [http://www.genomics.agilent.com/primerDesignProgram.jsp primer design tool]. Account required. | |||
* Use guidelines in the manual: | |||
** [[image:Quik_change_lightning_multi_kit_primer_instructions.jpg|thumb|upright=2.0|center|primer design instructions from the [http://www.chem.agilent.com/library/usermanuals/Public/210514.pdf manual]]] | |||
== Reaction Recipes == | |||
* '''Regular''' (2 reverse compliment primers) | |||
* '''Lightning''': | |||
** [[image:Quik_change_lightning_multi_kit_reaction_recipe.jpg|thumb|upright=3.3|center|reaction recipe instructions from the [http://www.chem.agilent.com/library/usermanuals/Public/210514.pdf Lightning manual]]] | |||
** QuikSolution (DMSO) can be added: 0 - 0.75uL per 25uL of reaction. | |||
** ds-DNA template = 50ng per 25uL for <5 kb, or 100 ng per 25uL if for >5 kb. | |||
*** 2ng/uL works fine for 6kb plasmids. -'''[[User:Janet Matsen|Janet Matsen 9/2014]]''' | |||
** primers: | |||
*** our lab usually dilutes primers to 10uM, and the recipes are for ng. Usually 10uM is ~100ng/uL. | |||
== 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.) | ||
* 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 15:20, 16 September 2014
Back to Protocols
Manuals
- QuikChange II: use 2 oligos (reverse compliments) and only do mutations at one priming site
- QuikChange II Lightning: use 1 oligo, and can introduce mutations at multiple sites
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.
Primer Design
- Use online primer design tool. Account required.
- Use guidelines in the manual:
primer design instructions from the manual
Reaction Recipes
- Regular (2 reverse compliment primers)
- Lightning:
reaction recipe instructions from the Lightning manual - QuikSolution (DMSO) can be added: 0 - 0.75uL per 25uL of reaction.
- ds-DNA template = 50ng per 25uL for <5 kb, or 100 ng per 25uL if for >5 kb.
- 2ng/uL works fine for 6kb plasmids. -Janet Matsen 9/2014
- primers:
- our lab usually dilutes primers to 10uM, and the recipes are for ng. Usually 10uM is ~100ng/uL.
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.
