targeted random mutagenesis gave majorly CCCs - (Oct/16/2008 )

Hello,

I am trying to use fusion PCR and degenerative primers (primers having NNKs) to mutate 7 continuous amino acids. When I sequence individual clones, this 7aa region turned out to be mostly CCCCCCC…. The chromatograph of these positions looked like mixed peak instead of one distinct single peak. When I sequenced the whole PCR product, these positions look like mixed peaks but still give CCCCs because C peak is the highest.

Is this saying I am getting CCCs instead of random NNKs? Could this be sequencing artifact? Thanks!

Xin

It sounds as if you have not picked single colonies from your transformations. I would restreak for single colonies and then do minipreps and sequence on isolated colonies. You should get only one sequence per clone.