For a lab course we have been assigned to "remove a stop codon, using mutagenesis, that is in the middle of a gene in order for the full gene to be expressed. This will then fluoresce."
To do this we are considering site directed mutagenesis, which can replace the stop codon, however is this a good method? Are there any alternatives to SDM using Quikchange that are better? What are the pros and cons of the SDM we are thinking of performing? To see if we succeeded we will then use DNA sequencing and gel electrophoresis.
Our overall plan looks like this:
Gathering plasmids from E. coli
Gel electrophoresis of the plasmid DNA
- Perform the mutagenesis using Quikchange
- Transformation by heat-shock
- Use a lac promoter to produce the gene protein
One issue I could think of was, how do we make sure the primers carrying the mutation don't anneal to each other due to their complementarity?
Is overlap-extension a good alternative? Are there any advantages to using transposon-based or point-directed mutagenesis?