Site-directed mutagenesis (SDM) is a powerful molecular tool for studying the effects of DNA sequence changes on protein function. There are a variety of options and protocols available to perform SDM, including a number of kits to facilitate the process. The Quikchange II SDM Kit from Stratagene is an updated version of the original kit with improved PfuUltra High Fidelity DNA polymerase. The method relies on a DNA polymerase-catalyzed, mutagenic-primed reaction from your template plasmid, followed by digestion with Dpn I (digests only methylated bacterial DNA). The resulting DNA is transformed into XL1-Blue Supercompetent bacteria, which are grown overnight and analyzed for successful mutagenic incorporation.
The kit contains all the elements necessary to perform SDM, except your cloned DNA fragment and the primers that insert any base-pair changes. Reagents include PfuUltra High Fidelity DNA polymerase, 10X reaction buffer, dNTP mix, Dpn I restriction enzyme, QuikChange control plasmid and control primers, XL-1 supercompetent cells and the pUC18 control plasmid. Thorough guidelines are provided for designing your own mutagenic oligonucleotide pair (one for each DNA strand). Until recently, Stratagene had a great online tool for designing these oligos. They have now replaced it with a Tm calculator (http://www.stratagene.com/QPCR/tmCalc.aspx). Based on my experience, I would recommend following the design guidelines to obtain successful results. In particular, I would pay attention to oligo purification following synthesis, which calls for a FPLC or PAGE level of purity.
I have had 100% success with 10 different oligo sets and contructs following the guidelines, only having to optimize the oligo concentrations. These include anything from a standard one base pair change in a 5kb construct to multiple amino acid substitutions in constructs of over 10kb. My only problem with the kit is the limited volume of reagents based on applications, thus care is need when pipetting small volumes to ensure enough reagent for all 10 reactions. One feature that is particular useful in the kit is the positive control they provide, which is good the first time you perform SDM with this kit. To generate the control, the stop codon for the beta-galactosidase gene is removed from the control plasmid using a provided oligo, thus success results in blue colonies on X-gal/IPTG agar plates.
Overall, after trying many different SDM kits, I would recommend the Quikchange II SDM Mutagenesis Kit. The method is very quick, efficient and easy, even with basic knowledge of molecular biology thanks to the well-explained protocol.
Barry Hudson
Post-Doctoral Fellow
Surgical Science
Columbia University