Quikchange PCR dNTP mix? - (Nov/30/2007 )

Hello,
I am trying to do site directed mutagenesis based on the Quikchange principle but without actually buying the kit. The kit comes with a dNTP mix that it says is propprietary. Since I am not buying the kit, I have to use my own dNTP mix. How much should I use. I normally use 200micromolar for a 50 microlitre reaction.
Any ideas?

They have figured out that something has to be proprietary for them to make money on the kit. I suspect that what is proprietary is that there is nothing that is proprietary.

Use as much as you would for a normal PCR with the polymerase you're using. That's the way my colleagues and me are (succesfully) doing it all the time (with different polymerases depending on the length of the plasmid we do mutagenesis on).

As vairus suggested, we use the same dNTP concentration that we would use for normal PCR reactions.

Hi Vairus, thanks for the input. Do you have any suggestions on what polymerase to use for a 6.5 kb plasmid? I am trying with Phusion polymerase from Finnzymes, but the PCR is not working. Can you help?

I've used PfuTurbo and PfuUltra with good succes. Maybe you should lower your annealing temp (I've never used phusion, but people here seem pretty satisfied with it).

I used pfu turbo.

treat it like a normal PCR, and go through the same steps as you would trying to optimise a PCR.

V

I'm LOVING Phusion at the moment- it seems to be working great for everything that I'm doing (although now that I've said that everything will probably start failing!!)

What denaturation temp are you using??

Thanks everybody for all the great tips. I am using denaturing temperature of 98 C and annealing temp of 55 C and extension temp of 68 C.