The Insertion of Mutagenesis
Insertion mutagenesis, missing T base corresponds to first base of forward primer, any thoughts?
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.
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