Hello, 

I am conducting site directed deletion mutagenesis of pcDNA5 and I am acquiring my desired mutations, however, there is always an additional one or two codons deleted following my desired mutation (T or TT additional deletion). I'm using Q5 and Deep Vent from NEB, and still have this extra base pair deletion issue. I thought it might be a primer issue, so I re-ordered new primers and I'm still encountering the same problem. I've optimized my thermocycler conditions and cleaned up my template in case of contamination or some sort, and still the same issue persists. Additionally, taking into account the exonuclease activity these polymerases have, I've lessened their concentration in my reaction, increased my primer concentration, and lessened extension time to avoid oligo "chewing" by the enzymes. The TT being deleted corresponds to end end of the common forward primer used in my  PCR reaction. I have a series of 5 different Reverse primers that I'm using to create these constructs, and have acquired 2 correct constructs out of the 5, but the remaining three posses the same TT or T deletion issue among their specified deletion regions. I've used all new solutions, fresh DNTPs, and buffer etc, and obtain good amplification, but these extra deletions at the deletion site of interest still persist. Any insight would be very helpful! 

Thanks so much!