Stratagene’s QuikChange II Site Directed Mutagenesis Kit is a simplified method to perform point mutations, change amino acids or delete/insert amino acids using a thermal cycling technique in combination with Dpn I digestion. QuikChange II replaces Stratagene’s original QuikChange Kit, although the only difference is the replacement of PfuTurbo DNA polymerase for PfuUltra high-fidelity DNA polymerase. Stratagene claims the change eliminates incidences of second site errors. Our lab did not find second site errors to be a significant concern with the previous kit; however Stratagene’s attemps to help reduce error is appreciated.
The kit procedure begins with your gene of interest in a double strand vector, purified simply using a plasmid-prep kit (Qiagen, in our case). Two primers, both containing the desired mutation, cover the area where the mutation is to be made. A detailed set of instructions are contained in the protocol for primer design and, in our experience, they are worth following. In particular, it is important to keep the desired mutation towards the middle of the primer and have them purified by FPLC or PAGE when ordering. We have typically used PAGE purification with good results.
The protocol begins with a series of different concentrations of your dsDNA (Stratagene recommends 5, 10, 20 and 50ng) to which the various components of the kit are added, including your primers at 125ng/reaction. Lastly, 1ul of PfuUltra is added and the reaction mixture is cycled in a PCR thermal cycler. The number of cycles depends on the procedure (e.g. 12 cycles for a point mutation; 16 for a single amino acid change, etc.). After this step, the reaction is cooled below 37oC and 1 ul of Dpn I restriction enzyme is added to each reaction and incubated at 37oC for 1hr. This step is the key feature to the QuikChange kit, as Dpn I enzyme is specific to methylated and hemi-methylated DNA. As such, DpnI digests the parental DNA template but does not digest the mutant-synthesized DNA. As most E.coli strains produce methylated DNA, they are not resistant to Dpn I digestion. Undigested DNA is then used to transform competent cells. The Stratagene kit comes with XL1-Blue supercompetant cells, which are require to be aliquoted specifically into Falcon 2059 polypopylene tubes prior to transformation. Transormed cells are incubated in NZY+ broth for 1hr in an orbital shaker and then plated LB-ampicillin agar plates with X-gal and IPTG, allowing for blue colony selection of positive mutants.
Stratagene’s QuikChange II Kit is a system that makes site directed mutagenesis an easy process. This is a rapid, one day procedure, which results in a high rate of positive mutants. We have used this kit to perform single point mutations predominantly, but also 2 and 3 mutations at one time with great success. A modification that we have made to the Stratagene protocol is to use Invitrogen’s TOP10 chemically competent cells instead of the provided XL1-Blue cells. These cells come pre-aliquoted with SOC media and this adds an extra level of convenience to the procedure. We follow Invitrogen’s protocol for transforming these cells and plate onto LB-Amp agar plates. Although the blue selection of positive colonies is lost, screening no more than 5 colonies invariably results in identification of positive mutants. This procedure is fast, simple and the most difficult part of the whole procedure is the design of the primers.
Senior Research Scientist
Stratagene's QuikChange II Site Directed Mutagenesis Kit
Fast, Simple, well designed kit. Has a very high rate of successful mutation. No special treatment required to template dsDNA.
Non aliquoted competent cells add extra step and time to procedure. Primer design can be tricky.
The Bottom Line
This kit is an excellent resource with almost guaranteed success in a very short time. There is very little competition for this kit on the market at the moment. If only all kits were as good as this!