I have degenerative primers for a gene that occurs upto 8 times in my genome. Each copy of the gene has varying specificity to the primers resulting in the dominance of a couple of products using my current method...but I want to amplify all or at least most of the genes.
Does anyone have any suggestions for decreasing the specificity of primers?
I am considering things like:
- Increase annealing duration
- Decrease annealing temp.
- Put in additives
- Remove additives (I'm currently using DMSO)
- Use normal Taq not HotStart Taq
- OR (as a last resort) design two set of primers to target a smaller group of genes
Does anyone have expereince with these option working or not working, as the case maybe?