NanoBio: Site-Directed Mutagenesis

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Site-Directed Mutagenesis

This protocol describes how make deletions, mutations, and additions to an existing plasmid. Site-directed mutagenesis is frequently used to remove unwanted BioBrick or BioFusion restriction sites from a part's sequence. I recommend using QuikChange's MultiSite Directed Mutagenesis Kit to do site-directed mutagenesis, even if only one site is to be mutated.

Materials

  • QuikChange Multi Site Directed Mutagenesis Kit (Stratagene, catalog # 200515)
  • template DNA
  • mutagenesis primers (1 per region to be changed)

Procedure

  • Design and order PAGE-purified primer(s). Stratagene's website has a nice program to help you design primers. Briefly, your primers must:
    • be between 25-45 bp long
    • have the site to be mutated approximately in the middle of the primer, with 10-15 bp flanking this site
    • have a melting temperature greater or equal to 75C
    • have a GC content of at least 40%
    • (only if using multiple primers): all bind to the same template strand
  • See the instruction manual for additional details.
  • If this is the first time you're doing this procedure, do the positive control included in the kit.
  • When doing the site-directed mutagenesis, follow the instructions in the manual, with these changes:
    • Do two pcr reactions: one with 1.0 uL of template DNA, and a second with 0.5 uL template DNA.
    • Digest with DpnI for at least 5 hrs., or preferably overnight, rather than for 1 hour.
    • XL-10 Gold Cells can be transformed in eppendorf tubes using SOC media, rather than in Falcon tubes with NZY+ media.
    • After adding the DpnI-digested pcr reaction to the XL-10 Gold cells, incubate on ice for 45 min - 1hr, instead of 30 min.
    • From 500 uL of SOC + transformants, plate 50uL. Centrifuge the remaining cells and media. Leaving the pelleted cells intact, remove all but ~150 uL of media. Re-suspend the cells, then spread them on a second plate. Essentially, you are plating all the cells from remaining 450 uL transformation mixture, but just decreasing the volume that you are spreading on the plate to ~150 uL.
  • In our experience, this procedure works well. There are other protocols around if you run into problems.
  • CAjoF 17:59, 31 March 2008 (EDT):.
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