Lidstrom:QuikChange Site-Directed Mutagenesis: Difference between revisions
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== The zillion kits == | |||
{| border="1" style="border-collapse:collapse;" | |||
|- | |||
! Kit | |||
! PCR: min/kb | |||
! # of cycles | |||
! Dpn1 digetstion time | |||
! Mutations @ multiple sites? | |||
! Manual link | |||
! 10 reaction kit item # | |||
! 30 reaction kit item # | |||
|- | |||
| QuikChange (original kit: don't buy) | |||
| 1 min? | |||
| | |||
| 1 hr | |||
| one pair of primers --> mutations in one DNA region | |||
| | |||
| [http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange/?cid=AG-PT-175&tabId=AG-PR-1160&searchText=200519 200519]. $263. | |||
| [http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange%20II/?cid=AG-PT-175&tabId=AG-PR-1161&searchText=200521 200518]. $700 | |||
|- | |||
| QuikChange II | |||
| 1 min | |||
| 12-18 | |||
| 1 hr | |||
| one pair of primers --> mutations in one DNA region | |||
| [http://www.chem.agilent.com/library/usermanuals/Public/200523.pdf QuikChange II manual] | |||
| [http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange%20II/?cid=AG-PT-175&tabId=AG-PR-1161&searchText=200523 200523]. $263 | |||
| [http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange%20II/?cid=AG-PT-175&tabId=AG-PR-1161&searchText=200524 200524]. $700 | |||
|- | |||
| '''QuikChange Lightning''' | |||
| 30 seconds | |||
| 18 | |||
| 5 minutes | |||
| one pair of primers --> mutations in one DNA region | |||
| [http://www.chem.agilent.com/library/usermanuals/Public/210518.pdf Catalog # 210518 (10 reactions) and #210519 (30 reactions), Revision E.01] | |||
| [http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange%20Lightning/?cid=AG-PT-175&tabId=AG-PR-1162&searchText=210518 210518]. $253 | |||
|[http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange%20Lightning/?cid=AG-PT-175&tabId=AG-PR-1162&searchText=210519 210519]. $673 | |||
|- | |||
| '''QuikChange MULTI''' | |||
| 30 sec | |||
| 30 cycles | |||
| 1 hr | |||
| '''YES'''. priming w/ multiple primers & primers aren't paired w/ reverse compliment | |||
| [http://www.chem.agilent.com/library/usermanuals/Public/200514.pdf manual: catalog # 200514 and #200515] | |||
| Academic: [http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange/?cid=AG-PT-175&tabId=AG-PR-1160&searchText=200515 200515]. $370 | |||
|[http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange/?cid=AG-PT-175&tabId=AG-PR-1160&searchText=200514 200514] = academic kit = $985. | |||
|- | |||
| '''QuikChange Lightning MULTI''' | |||
| 30 seconds | |||
| 30 cycles | |||
| 5 minutes | |||
| '''YES'''. priming w/ multiple primers & primers aren't paired w/ reverse compliment | |||
| [http://www.chem.agilent.com/library/usermanuals/Public/210513.pdf Manual for Catalog # 210513 and #210515] | |||
|Academic: [http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange%20Lightning/?cid=AG-PT-175&tabId=AG-PR-1162&searchText=210515 210515]. $357 | |||
| Academic: [http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange%20Lightning/?cid=AG-PT-175&tabId=AG-PR-1162&searchText=210513 210513]. $948. | |||
|- | |||
| QuikChange, XL (large templates) | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| [http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange/?cid=AG-PT-175&tabId=AG-PR-1160&searchText=200517 200517]. $263. | |||
| [http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange/?cid=AG-PT-175&tabId=AG-PR-1160&searchText=200516 200516]. $700 | |||
|- | |||
| QuikChange II, XL (large templates) | |||
| | |||
| | |||
| | |||
| one pair of primers --> mutations in one DNA region | |||
| [http://www.chem.agilent.com/library/usermanuals/Public/200521.pdf XL manual] | |||
| [http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange%20II/?cid=AG-PT-175&tabId=AG-PR-1161&searchText=200521 200521]. $263. | |||
| [http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange%20II/?cid=AG-PT-175&tabId=AG-PR-1161&searchText=200522 200522]. $700 | |||
|- | |||
| QuikChange II + Electroporation cells (not chem. comp) | |||
| | |||
| | |||
| | |||
| one pair of primers --> mutations in one DNA region | |||
| [http://www.chem.agilent.com/library/usermanuals/Public/200555.pdf QC + electroporation manual] | |||
| [http://www.genomics.agilent.com/en/Site-Directed%20Mutagenesis/QuikChange%20II/?cid=AG-PT-175&tabId=AG-PR-1161&searchText=200555 200555] | |||
| | |||
|} | |||
== 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/210513.pdf QuikChange II Lightning]: use 1 oligo, and can introduce mutations at multiple sites | |||
==QuikChange Basics == | |||
* You can buy a kit from Agilent or [[Richard_Lab:Site_Directed_Mutagenesis|use your own ingredients]] (not true for multi kit though). | |||
* 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 [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 == | |||
* '''Lightning non-multi''' (2 reverse compliment primers) | |||
** [[image:Quik_change_lightning_NOT-multi_kit_reaction_recipe.jpg|thumb|upright=3.3|center|reaction recipe instructions from the [http://www.chem.agilent.com/library/usermanuals/Public/200523.pdf non-multi Lightning manual]]] | |||
* '''Lightning Multi''': | |||
** [[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/210513.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. | |||
*** Add ~0.5uL of primer per 8uL of reaction. *'''[[User:Janet Matsen|Janet Matsen 9/2014]]''' | |||
== Thermocycling == | |||
*'''Regular LIGHTNING Kit''' (but not multi LIGHTNING kit) | |||
** [[image:Quik_change_lightning_NOT_multi_kit_thermocycling.jpg|thumb|upright=3.3|center|thermocycling protocol from [http://www.chem.agilent.com/library/usermanuals/Public/210518.pdf NOT-multi manual]]] | |||
*'''Multi Lightning Kit''' | |||
** [[image:Quik_change_lightning_multi_kit_thermocycling.jpg|thumb|upright=3.3|center|thermocycling protocol from [http://www.chem.agilent.com/library/usermanuals/Public/210513.pdf Multi + Lightning manual]]] | |||
== 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 [[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. |
Latest revision as of 15:28, 22 September 2014
Back to Protocols
The zillion kits
Kit | PCR: min/kb | # of cycles | Dpn1 digetstion time | Mutations @ multiple sites? | Manual link | 10 reaction kit item # | 30 reaction kit item # |
---|---|---|---|---|---|---|---|
QuikChange (original kit: don't buy) | 1 min? | 1 hr | one pair of primers --> mutations in one DNA region | 200519. $263. | 200518. $700 | ||
QuikChange II | 1 min | 12-18 | 1 hr | one pair of primers --> mutations in one DNA region | QuikChange II manual | 200523. $263 | 200524. $700 |
QuikChange Lightning | 30 seconds | 18 | 5 minutes | one pair of primers --> mutations in one DNA region | Catalog # 210518 (10 reactions) and #210519 (30 reactions), Revision E.01 | 210518. $253 | 210519. $673 |
QuikChange MULTI | 30 sec | 30 cycles | 1 hr | YES. priming w/ multiple primers & primers aren't paired w/ reverse compliment | manual: catalog # 200514 and #200515 | Academic: 200515. $370 | 200514 = academic kit = $985. |
QuikChange Lightning MULTI | 30 seconds | 30 cycles | 5 minutes | YES. priming w/ multiple primers & primers aren't paired w/ reverse compliment | Manual for Catalog # 210513 and #210515 | Academic: 210515. $357 | Academic: 210513. $948. |
QuikChange, XL (large templates) | 200517. $263. | 200516. $700 | |||||
QuikChange II, XL (large templates) | one pair of primers --> mutations in one DNA region | XL manual | 200521. $263. | 200522. $700 | |||
QuikChange II + Electroporation cells (not chem. comp) | one pair of primers --> mutations in one DNA region | QC + electroporation manual | 200555 |
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 (not true for multi kit though).
- 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:
Reaction Recipes
- Lightning non-multi (2 reverse compliment primers)
- Lightning Multi:
- 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.
- Add ~0.5uL of primer per 8uL of reaction. *Janet Matsen 9/2014
Thermocycling
- Regular LIGHTNING Kit (but not multi LIGHTNING kit)
- Multi Lightning 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.