Lidstrom:QuikChange Site-Directed Mutagenesis: Difference between revisions
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Back to [[Lidstrom:Protocols|Protocols]] | Back to [[Lidstrom:Protocols|Protocols]] | ||
== | == The zillion kits == | ||
{| border="1" style="border-collapse:collapse;" | {| border="1" style="border-collapse:collapse;" | ||
|- | |- | ||
! Kit | ! Kit | ||
! PCR: min/kb | ! PCR: min/kb | ||
! # of cycles | |||
! Dpn1 digetstion time | ! Dpn1 digetstion time | ||
! Mutations @ multiple sites? | ! Mutations @ multiple sites? | ||
! Manual link | ! Manual link | ||
! 10 reaction kit item # | ! 10 reaction kit item # | ||
| | |||
! 30 reaction kit item # | ! 30 reaction kit item # | ||
|- | |- | ||
| QuikChange (original kit: don't buy) | | QuikChange (original kit: don't buy) | ||
| 1 min? | | 1 min? | ||
| | |||
| 1 hr | | 1 hr | ||
| one pair of primers --> mutations in one DNA region | | one pair of primers --> mutations in one DNA region | ||
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| QuikChange II | | QuikChange II | ||
| 1 min? | | 1 min? | ||
| | |||
| 1 hr | | 1 hr | ||
| one pair of primers --> mutations in one DNA region | | one pair of primers --> mutations in one DNA region | ||
Line 30: | Line 34: | ||
| QuikChange MULTI | | QuikChange MULTI | ||
| 1 min? | | 1 min? | ||
| | |||
| 1 hr | | 1 hr | ||
| '''YES'''. priming w/ multiple primers & primers aren't paired w/ reverse compliment | | '''YES'''. priming w/ multiple primers & primers aren't paired w/ reverse compliment | ||
Line 38: | Line 43: | ||
| QuikChange Lightning | | QuikChange Lightning | ||
| 30 seconds | | 30 seconds | ||
| | |||
| 5 minutes | | 5 minutes | ||
| one pair of primers --> mutations in one DNA region | | one pair of primers --> mutations in one DNA region | ||
Line 46: | Line 52: | ||
| QuikChange Lightning MULTI | | QuikChange Lightning MULTI | ||
| 30 seconds | | 30 seconds | ||
| | |||
| 5 minutes | | 5 minutes | ||
| '''YES'''. priming w/ multiple primers & primers aren't paired w/ reverse compliment | | '''YES'''. priming w/ multiple primers & primers aren't paired w/ reverse compliment | ||
Line 54: | Line 61: | ||
| QuikChange II Lightning MULTI | | QuikChange II Lightning MULTI | ||
| 30 seconds | | 30 seconds | ||
| | |||
| 5 minutes | | 5 minutes | ||
| '''YES'''. priming w/ multiple primers & primers aren't paired w/ reverse compliment | | '''YES'''. priming w/ multiple primers & primers aren't paired w/ reverse compliment | ||
Line 64: | Line 72: | ||
| one pair of primers --> mutations in one DNA region | | one pair of primers --> mutations in one DNA region | ||
| 1 min? | | 1 min? | ||
| | |||
| 1 hr ? | | 1 hr ? | ||
| [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=200517 200517]. $263. | ||
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| QuikChange II, XL (large templates) | | QuikChange II, XL (large templates) | ||
| 1 min? | | 1 min? | ||
| | |||
| 1 hr? | | 1 hr? | ||
| one pair of primers --> mutations in one DNA region | | one pair of primers --> mutations in one DNA region | ||
Line 78: | Line 88: | ||
| QuikChange II + Electroporation cells (not chem. comp) | | QuikChange II + Electroporation cells (not chem. comp) | ||
| 1 min? | | 1 min? | ||
| | |||
| 1 hr | | 1 hr | ||
| one pair of primers --> mutations in one DNA region | | one pair of primers --> mutations in one DNA region |
Revision as of 18:07, 17 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? | 1 hr | one pair of primers --> mutations in one DNA region | QuikChange II manual | 200523. $263 | 200524. $700 | ||
QuikChange MULTI | 1 min? | 1 hr | YES. priming w/ multiple primers & primers aren't paired w/ reverse compliment | Academic: 200515. $370 | 200514 = academic kit = $886.50. | |||
QuikChange Lightning | 30 seconds | 5 minutes | one pair of primers --> mutations in one DNA region | 210518. $253 | 210519. $673 | |||
QuikChange Lightning MULTI | 30 seconds | 5 minutes | YES. priming w/ multiple primers & primers aren't paired w/ reverse compliment | 210514 & 210516 manual | Academic: 210515. $357 | Academic: 210513. $948. Non-academic # = 210514. | ||
QuikChange II Lightning MULTI | 30 seconds | 5 minutes | YES. priming w/ multiple primers & primers aren't paired w/ reverse compliment | Academic: 210515. $357 | Academic: 210513. $948. | |||
QuikChange, XL (large templates) | FAST (30 sec/kb PCR, "5 min" Dpn1) | one pair of primers --> mutations in one DNA region | 1 min? | 1 hr ? | 200517. $263. | 200516. $700 | ||
QuikChange II, XL (large templates) | 1 min? | 1 hr? | one pair of primers --> mutations in one DNA region | XL manual | 200521. $263. | 200522. $700 | ||
QuikChange II + Electroporation cells (not chem. comp) | 1 min? | 1 hr | 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
- Regular (2 reverse compliment primers)
- Lightning:
- 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.