site-directed pcr mutagenesis

Mikhail Sinev sinev at nospam.rci.rutgers.edu
Thu Oct 17 14:05:42 EST 2002


NTP concentration? Any idea.

ES

"D.K." <dk at no.email.thankstospam.net> wrote in message
news:aol6lk$qr$1 at news.doit.wisc.edu...
> S <rohrersusannenospam at hotmail.com> wrote:
> >
> >
> >jaderyan12 at yahoo.com wrote:
> >
> >> I'm trying to introduce a mutation at the 470aa of a 500aa protein.
First I
> > made the 1400bp base and the 100bp piece but trying to get them to
generate
> > the full length gene is proving impossible.  I have tried using 4-12
cycles of
> > annealing without primers, then adding the primers and continuing with
30 more
> > cycles.  I have tried temperatures from 50-60.  Does anyone have any
onther
> > suggestions?
> >
> >Stratagene's quickchange kit? (no endorsement except that I know it
works)
> >
> >Crossover PCR might work  for a homemade protocol (actually that's what
you
> > appear to be doing)
>
> There is nothing wrong with homemade Quikchange either. It works exactly
> the same way. The kit is just Pfu Turbo + buffer + competent cells
> (home made electropration cells are better anyway) + dNTPs + DMSO (for
> an XL version).
>
> DK





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