QuikChange Mutagenesis Protocols

QuikChange mutagenesis is the brand name of a kit used in biotechnology research to induce genetic mutation or DNA alteration. Agilent Technologies is the company behind the product, which is available in two versions, QuikChange II and QuikChange Lightning. Both kits contain a buffer solution, the enzyme DNA polymerase, color reagents, DNA sequences or primers, and cell samples.

Mutant Strand Synthesis Reaction Protocol

• Technicians synthesize two oligonucleotides or short DNA sequences containing the desired mutation. Then they prepare the control reaction with the DNA sequences, dNTP mix solution and DNA polymerase enzyme, adding distilled water. Later, they prepare a series of sample reactions using various concentrations of double-stranded DNA (dsDNA), while keeping primer or oligonucleotides concentration constant, according to the manufacturer's instructions. They keep the samples at 203 degrees F for 30 seconds. The sample containing DNA segment number two is also tested under temperature cycling, a technique where temperature changes during short periods of times. Finally, the samples are placed on ice for two minutes.

Dpn I Digestion of the Amplification Products Protocol

• Dpn I restriction enzyme cuts the DNA at specific sites. One micro-liter of this enzyme is added directly to each amplification reaction below its standard mineral oil overlay using a micro-pipette or specialized aerosol-resistant pipette tip. However, a mineral oil overlay is only required if the thermal cycler, an apparatus used to amplify DNA segments, does not have a hot-top assembly. The reaction is gently mixed and later put in a micro-centrifuge for one minute. After that, the samples are incubated at 98.6 degrees F for one hour.

Transformation of XL1-Blue Supercompetent Cells Protocol

• XL1-Blue is a tetracycline-resistant cell line that comes with the QuikChange Mutagenesis kits. During this protocol, technicians thaw the cells, which were previously kept at -112 degrees F, on ice. Then they add one micro-liter of the Dpn I-treated DNA, heat it to 107.6 degrees F for 45 seconds and place the reaction on ice for two minutes. After this procedure, technicians add a preparation of NZY (casein hydrolysate and yeast extract) preheated to 107.6 degrees F and incubate for one hour. The preparation is then put on agar gel plates and incubated at 98.6 degrees F for 16 hours.