Hello,
I need to change two adjacent nucleotides.
* I've added 5% DMSO
* I've used an annealing temp that is 5 degrees higher than the tm of the primers.
* Im using a hot start kit
But after sequencing is still get a long repeat of the primer in the site of mutagenesis.
Analysis show that the dimerization in due to nucleotides that are adjacent to the mutations, so I cant design a primer without them.
What else can I try?
Do you think its possible to use a long primer with the mutation flanking? A bit like Gibson reaction?
Thank you!