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?
SDM will be the perfect tool for this mission, unless the plasmid in question is very large (20+ kb).
When you have performed the SDM, digest the products with dpnI to remove the original plasmids, transform e.coli and use a selective media matching your plasmid. Only bacteria with the plasmid will then be able to survive and form colonies, pick several colonies and perform a miniprep for each and sequence the area of interest. You can use the rest of the miniprep with the correct plasmid for transformation and expression. So skip step 5.
The 'Quickchange' method is probably what you want. If you are worried about self complementary (which you shouldn't be too much) you can actually stagger the primers so they are not fully complementary. only the 10bp need to overlap where the mutation is. I included a small diagram below where the X marks the base you want to change
One alternative, which I use more than the SDM is by using TypeIIS restriction enzymes. This way you can mutate as many bases as you want in one go! See this article