Quick Change mutli kit

M Sin esineva at netscape.net
Mon Dec 8 14:49:35 EST 2003


Could you please provide dNTP concentration you use for ""Single" QuikChange
"?


"D.K." <dk at no.email.thankstospam.net> wrote in message
news:vs2osvsb4d2b4ddbrtdtlri0ci34q8as12 at 4ax.com...
> On 1 Dec 2003 19:59:23 -0000, Laura.Segall at mail.mcgill.ca (Laura Segall)
wrote:
>
> >I was wondering if anyone can help me out.
> >
> >I am trying to introduce several mutations into a plasmid (6Kb) using
> >the stratagene quick change multi-site kit.  All primers were designed
> >as suggested in the kit manual, but I obtained no PCR product.  Template
> >was checked on a gel prior to PCR, primers were phosphorylated, cleaned
> >on a column and quantified.  Still no product.  Annealing temp was
> >dropped to 52C (from 55C), still no product.  Primer concentration was
> >fiddled with, still no product.  Technical assistance suggested
> >transforming the bugs anyway, saying that the PCR yield may simply be
> >too small to see on a gel.  Personnally, I prefer to see a product on a
> >gel first.  Other than that, they had no suggestions.   I am trying the
> >transformation tonight and will fiddle with the ratios of
template:primer.
> >
> >Any help/suggestions would greatlty be apreciated.Getting a little
> >frustrated here.
>
> [Probably not the suggestion you are looking for :-)]
>
> Personally, I find "multi" rather finicky. In my experience, on average
> it is always faster to do two sequential "simple" QuikChanges than to
> make it work in every case.
>
> "Single" QuikChange never fails for me - I simply always do first 3
> cylces with the two primers separately, then mix the two reactions and
> do another 15 cycles (orignally described in Biotechniques some years
> ago). Besides codon changes, did 33 bp insertions and ~ 600 bp deletions.
> And we don't buy any kits, only polymerase (Pfu Ultra - works just as
> well as Turbo and gives less errors). Even mediocre home-made
> electroporation "competent" cells are better than Stratagene's
> competent cells than come with the kit. Besides polymerase and cells,
> the kit contains absolutely nothing useful.
>
> DK
>





More information about the Methods mailing list