site-directed pcr mutagenesis

[D.K.]

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