I have done site-directed mutagenesis with pALTER-II from Promerga.
In reaction system amp repair oligos and my mutagenic oligos are
included. I am not quite sure whether mutagenic oligos sticked to
ssDNA template or not . The reason that I suspected this is that I
once did one time mutagenesis in which template DNA contains wrong
orintation cloned insert gene, that means the insert in ss DNA is not
complementary to the mutagenic oligos, but I still got the synthesized
dsDNA and transformed the cells successfully. Certainly, the
synthesized dsDNA is initiated by amp repair oligos not by both amp
oligos and mutagenic oligos. So, I am not sure whether this time
(insert dna is in right orintation) synthesized dsDNA is initiated
by only repair oligos or by both. How can assess the possibility of
mutagenic oligo binding to template DNA before I do the sequence
analysis if I don't set up a control reaction system containing only
mutagenic oligos as a primer ( Promerga manual doesn't suggest this
type of control system)?
Does anyone there has ideas about this ?