NanoBio: Site-Directed Mutagenesis

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
 * Note: Use Stratagene's Tm calculator to determine the Tm of your primers; it is different from Vector NTI's calculated Tm.
 * 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):.