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Site-directed mutagenesis

wliu at receptor.pharm.uwa.edu.au wliu at receptor.pharm.uwa.edu.au
Mon Oct 14 05:01:06 EST 1996

Hi, Netters,
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 ?


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