Are u really trying to delete the codon or mutate it?. Mutating it would be easier, in case your template is tricky. For example, In my hands pcDNA3 was a difficult to mutate backbone (I found online references that others also had similar experiences). If u have not tried SDM with the same backbone vector before, this could be a reason.

Phusion works excellent for site-directed mutagenesis and deletions. Here is the protocol I'm using:

http://openwetware.org/wiki/User:Maximilian_Peters/site-directed-mutagenesis

Hi,

first I have a few question: What is your problem? Do you have colonies or not? If you have colonies, are all of them wildtype? Or do they carry some ofter mutations?

Second: I also have already some suggestions: i) I would check the primer again, maybe there is already a mistake. I never used special programms for this, I "designed" them by myself and it works fine. ii) I would try a gradient PCR, means try several annealing temperatures, from 50-70°C. You can mix all these reaction and can go ahead. iii) Use DMSO in the PCR. iv) Use less template, make sure, that DpnI digest is complete. v) What is the result of removed codon? Maybe it will affect your organism, sometimes expression is leaky, you might end up with a toxic protein, your organism will try to survive and starts mutating itself (happens to me a while ago, and finaly it was not possible to mutate the gene at all).

With pHUSION I always add 1-7 ul of DMSO to the reaction, this will help if you have any hair pin structures in your primers.DMSO can be used with pfu as well. Also do not do more than 25 cycles with pHUSION (mutagenesis is usually about 18 cycles).

If mutagenesis still doesn't work try doing an inverse PCR deleting the stop codon, (at least you should be able to see the product on a gel) you can design the primers to have homologous ends and then recombine the PCR product using In-fusion (clontech) rather than ligase.

Hi Itzia,

Did you analyze some of your PCR on a gel to determine if you have any product?

If you see a lot of primer dimer formation on your gel and no product, that could be solved with some DMSO or a longer denaturation step. Your reaction recipe looks fine - you can use less primer (2.5 - 4.0 uM final concentration) and less template (50 ng).

I would not proceed with the DpnI digest or transformation until you've taken a look at the PCR products. By the way, the kit is also unnecessary for site-directed mutagenesis.