The StarGate Combi Entry Cloning Set also allows the site-directed mutation of the gene of interest - within a sequence stretch of up to 80 bases in length - during generation of the Donor Vector.
The only difference in the site-directed mutation procedure is that two PCRs have to be performed instead of a single PCR whereby the gene internal PCR primers introduce the desired nucleic acid sequence changes (see Figure on right hand side).
The design of the essential primers is performed using IBA’s easy-to-use software "StarPrimer D’Signer" coming with the set.
To design the primers the sequence of the gene of interest (GOI) as present in the template as well as the desired mutated gene sequence simply have to be pasted into the respective StarPrimer D’Signer sequence windows. The software displays the sequence of the desired primers, which can then be ordered at the IBA's webshop.
Once PCR has been performed using the StarPrimer D’Signer developed primers, both PCR products are mixed with pENTRY-IBA and StarCombinase and are incubated for one hour. After transformation, E. coli clones harbouring the desired Donor clone with the mutated GOI can be easily detected using blue/white selection.
The StarPrimer D’Signer does not only help with the primer design, it will also guide you step by step through the whole PCR process.
|
