Lidstrom:QuikChange Site-Directed Mutagenesis
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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 = $886.50.|
|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|
- 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
- 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 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.
- Use online primer design tool. Account required.
- Use guidelines in the manual:
- 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
- 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
- Regular LIGHTNING Kit (but not multi LIGHTNING kit)
- Multi Lightning Kit
- 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.