Frank O. Fackelmayer Frank at Fackelmayer.de
Wed Jul 4 09:23:32 EST 2001

Soren Rasmussen wrote:
> Can you tell me the concentration of dNTP in the Stratagene QuikChange
> site-directed mutagenesis kit #200518? Somehow that has been used up now,
> but we still have many reactions left. Stratagene keeps the composition of
> the dNTP as a secrete.

I cannot give you the concentrations in the original stratagene kit, but
we have used the same principle with dNTP concentrations just in a
conventional PCR. In our case, this is 0.1mM of each dNTP. Works as
expected, no problems. With Pfu polymerase, fidelity is not a major
concern anyway. In most cases, more than 80% of analysed clones have the
right mutation.

We found it helpful to run more cycles (25-30), digest with DpnI for a
longer time (2h), and use really good competent cells. Usually, the
mutagenesis is sucessful when you can detect a PCR product of the
expected size after PCR and DpnI digest. Reactions with no detectable
product use to be bad and are not worth transformation into bacteria.
Optimize the PCR until you get product.


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