Quick Change mutli kit
no.email at thanks.to.spam.net
Tue Dec 9 09:42:12 EST 2003
In article <3fd4d608$1 at rutgers.edu>, "M Sin" <esineva at netscape.net> wrote:
>Could you please provide dNTP concentration you use for ""Single" QuikChange
I always use 0.2 mM in QuikChange.
BTW, I found the original reference for that approach:
BioTechniques, 26(4):680-2, 1999
>"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)
>> >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
>> >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.
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