Ahmet Alptekin Hi, I saw your other post as well. I'm sorry you had this T missing in your mutagenesis experiment. Because the changes looks consistent I would assume is not a sequencing error although it's always good to get an unequivocal reading. -

Using the Q5 from NEB is pretty much the same with my approach of using custom made 5' phosphorylation. In Q5 you use KLD (kinase, ligase, Dpn1). In my case I don't deal with kinases, the primers are ordered, at a larger mass, are more expensive, but usually I request SDS-PAGE purification or the best purification method they have (FPLC once), the amount of primer you receive is much less than what you ordered because the amount will decrease in the purification step but whatever you receive is likely to be correct.

So I do the inverse PCR with a high fidelity polymerase like Pfu, purify, ligate, Dpn1. Transformation. It's usually very robust.

In your case, since you already have the Q5 kit, I would repeat the experiment but reorder the primers with a purification step, it will be more expensive but not as expensive as if asking for a 5'-phosphorylation. It should work because the primers are bound to be correct. But other options are good too. Good luck.

I agree with Oscar, the problem probably lies with the oligo. I would also reorder the oligo, with a purification step (e.g. HPLC or gel-purified). However, even with a low grade oligo, i.e. desalted only, it is a bit strange that all of the clones you tested should be missing the first base of the primer. The primer must have been of quite poor quality indeed, unless it was degraded due to inadequate storing conditions: keep stocks of oligos frozen at high concentration (100 µM) in Tris-HCl 10 mM + 1 mM EDTA and make a fresh dilution in water before use.

You are using a primers with mistake that make the error , you should test these primers through purifying using gel electrophoresis