I attempted to use Overlap PCR to introduce two mutations (F to A mutation) into the DNA sequence of a protein im studying. The mutations were carried on two seperate primers i.e. one on primer B and the second on primer C. PCR products AB and CD were the correct size when checked on a gel. Again the final product was the expected size.
However after sending the final PCR product away for sequencing, the results i recieved back indicated that no mutations had been introduced and after a blast was proformed it was a 100% identical to the wild-type proteins DNA sequence.
Can anyone help me understand why that happened?
and what i can do troubleshoot this?
would appreciate it