Now that custom gene synthesis costs have made ordering a DNA plasmid with any desired sequence extremely affordable, it is difficult to see the importance of teaching young molecular biologists how to design primers for site directed mutagenesis, adding tags, or C-terminal truncations. I recall during my PhD how our lab’s molecular biology guru tried to teach me how to design mutagenesis oligos to introduce a novel restriction site and how important the GC-clamp was.
PrimerGenesis is a website that designs mutagenesis oligos for you. Other functionalities of the online tool include primer design for inserting tags, and C-terminal truncation mutants. Tools for calculating solution concentrations (remember CV=C’V’), protein molecular weights and isoelectric points are also available as part of this free online site.
The general workflow for using this web-based tool is fairly straightforward. For example to generate point mutants, after entering the coding sequence of a gene and the vector sequences preceding and following the coding sequence, the user must indicate with dropdown menus which mutation will be made. The website then proposes a variety of mutagenesis primers and indicates the melting temperatures for each. Once the user orders the primers, a Quick PCR Cloning Kit can be used for ligation-free cloning.
The main argument for outsourcing custom plasmid generation is that the time a researcher spends designing primers, optimizing PCR, purifying restriction digests, ligating and picking colonies is excessive compared to the relatively low cost of paying a company to make a sequence-perfect plasmid. PrimerGenesis will undoubtedly reduce the amount of time researchers spend making their own plasmids and will be a good resource for molecular biologists in training.