quikchange question

Rani Maurice Geha geha at hsc.usc.edu
Mon Mar 24 16:19:20 EST 1997


In article <5grpjp$bhg at eldborg.rhi.hi.is>, thorarb at rhi.hi.is (Thorarinn
Blondal) wrote:

> I'm curious about quikchange mutagenesis kit from stratagene.
> has anyone experince concerning the kit?  Is it as fast and accurate 
> as advertized?  What is the max size of plasmid it can mutate?
> 
> With graditude  Th.Blondal
>                 &
>                 A.Palsson
>                 elm:ap at lif.hi.is


I bought quickchange as soon as it came out, and at first I was not able to
get any successful mutations, even with the kits own positive control and
following the protocol to the letter. Latter, I simply lowered the
annealing temperature from 55C to 52C and I was able to obtain many
colonies with an efficiency of about 80 percent.

Yes, it is as fast and accurate as advertized. You simply mix the plasmid
with the primers, the dNTP mix, and Pfu DNA pol and do 12 thermal cycles
(about 4 hours), then transform into the provided Supercompetent XL1-Blue
cells and the next day you have your mutants. The Pfu pol has very high
fidelity and since your doing only 12 cycles there is little chance of
error (see NAR 1996, 24:3546-3551).

I don't know what the max plasmid size is, they don't say. But I use an 8.5
kb plasmid. It is the easiest kit I have used.

Rani Geha
graduate student
University if Southern California



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