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