The QuikChange® II XL Site-Directed Mutagenesis Kit by Stratagene is used for in vitro
site-directed mutagenesis of large targets. The kit can be used to make point mutations, replace amino acids, and insert or delete one or several amino acids. The kit follows a three step procedure, whereby PCR is used to introduce a mutation, the template DNA is digested away with the use of Dpn I
enzyme, and finally, the mutated molecule is transformed into ultracompetent cells.
The advantage of using this kit is that it comes with all the necessary reagents for performing the mutation/PCR reaction as well as XL10- Gold Ultracompetent cells, and control plasmids and primers. Also, this kit can be used for large targets, greater than 8 kb. No unique restriction sites or multiple transformations are required.
In order to start the procedure, you will need to design primers to introduce your desired mutation. This is the most critical step in the procedure and any deviation from the recommended guidelines will severely hinder your success. There are very clear guidelines for designing primers in the kit brochure, so be sure to follow them. In addition, the Stratagene website has a helpful QuikChange® Primer Design Program for your convenience. It is important that the desired mutation is in the middle of the primer sequence and that the primers be purified via FPLC or by PAGE. We have typically used PAGE purified primers with success. For primers longer than 45 base pairs, there is an increased chance of secondary structure formation, which may lower the efficiency of the reaction. We have found it helpful to aliquot a small amount of primer, then boil the aliquot at 95ºC for 2 minutes to denature any possible secondary structure prior to PCR.
Once your primers have been designed, a PCR reaction is performed with a special high fidelity polymerase, Pfu Ultra™ DNA Polymerase at 2.5 U/uL. There are specific guidelines for running the thermocycling reactions, as only 18 cycles are needed. Running more than 18 cycles risks introducing extraneous mutations into the plasmid.
The Dpn I restriction enzyme (10 U/uL) is then added to the PCR product for 1 hour to digest the parental DNA. Dpn I digests hemimethylated and methylated DNA, which includes most E. coli DNA, except for plasmid DNA isolated from dam- strains, such as JM110 and SCS110 (not compatible with this kit). Once the digest is complete, a simple transformation procedure is performed and colonies should appear overnight on selective media. The XL10-Gold cells, which are both recombination deficient (recA) and endonuclease deficient (endA1), have the phenotype Hfe, allowing an increased efficiency of larger DNA plasmid transformation.
Our only real complaint would be that, although we were able to obtain mutant plasmids through this kit, we did obtain a relatively high background of colonies. Typically, we had to sequence several clones to find the one we wanted.
A huge advantage of this kit is its speed. The entire procedure can be done in one day, and colonies can be obtained the next day, making it a very efficient and quick process. We would recommend this kit; however, the price is high, so be sure your primers are well-designed, according to the kit description, to ensure success.