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 = $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 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:
    • primer design instructions from the manual
      primer design instructions from the manual

Reaction Recipes

  • Lightning Multi:
    • reaction recipe instructions from the Lightning manual
      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.
    • 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)

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.
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